1. Art and Diseño

From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
Analysis of Major Histocompatibility Complex Class I Expression on
Reed-Sternberg Cells in Relation to the Cytotoxic T - c e l l Response in
Epstein-Barr Virus -Positive and -Negative Hodgkin’s Disease
By J.J. Oudejans, N.M. Jiwa, J.A. Kummer, A. Horstman, W. Vos, J.P.A. Baak, Ph.M. Kluin, P. van der Valk,
J.M.M. Walboomers, and C.J.L.M. Meijer
To get insight into the failure of the immune system to eradicate Epstein-Barr virus (EBV) harboring Hodgkin and ReedSternberg cells (H-RS cells), expressing the latent membrane
protein 1 (LMPl), we analyzed major histocompatibility
complex (MHC) class I expression on H-RS cells in relation
to the presence of activated cytotoxic cells, ie, granzymeB-expressing lymphocytes. H-RS cells in EBV+ cases of
Hodgkin‘s disease (HD) were found to express significantly
higher levels of MHC class I heavy- and light-chain molecules
compared with EBV- HD cases. When low levels of MHC
class I expression were found (mainly in EBV- cases), these
were not associated with low levels of the transporter protein associated with antigen presentation 1 (TAP-1). The rel-
atively high levels of MHC class I expression in H-RS cells in
EBV+ HD cases were accompanied by significantly higher
numbers of activated cytotoxic T lymphocytes (CTLs) as
shown by the presence of increased numbers of CD8 and
granzyme B+ lymphocytes. However, these cells were only
sporadically detected in the close vicinity of the H-RS cells.
These data suggest that mechanisms other than downregulation of MHC class I or TAP-1 expression on H-RS cells are
involved in the failure of the immune system to eradicate
EBV harboring H-RS cells. Probably, the function of activated
CTLs is locally inhibited by the H-RS cells or by reactive cells
in the vicinity of the H-RS cells.
0 1996 by The American Society of Hematology.
T
and natural killer (NK) cells. Recently, monoclonal antibodies (MoAbs) became available that react specifically with
g r a n ~ y m e s .These
’~
serine proteases are found in the cytoplasmic granules of CTLs and NK cells. Exocytosis of these
granules in the cytoplasm of the target cell will lead to
induction of DNA fragmentation and apoptosis of the target
ce~~.lc.16
Because expression of these proteins is restricted to
NK cells and activated CTLs.” antibodies directed against
granzymes are an excellent tool for in vivo detection of these
cells.
CTLs recognize endogenously processed antigens, such
as viral proteins and oncogene products, only when presented
at the cell surface as peptides bound to the MHC-I molecules.” A reduction or lack of MHC-I surface expression
can render aberrant cells resistant to CTLs, and may provide
a selective growth advantage for these cells. Such an immune
escape mechanism has been demonstrated for cervical carcinomas that harbor the human papilloma virus.” In these
carcinomas MHC-I downregulation was associated with the
loss of function of one of the transporter proteins associated
with antigen presentation (TAP- 1 ).2(’ Lack of peptide transport by TAP will inhibit peptide loading of empty class I
molecules, thereby severely reducing the complex stability.
Consequently, steady-state levels of MHC-I will be reduced.
A similar mechanism might be involved in the immune escape of EBV-harboring neoplastic cells in EBV’ cases of
HD.
The activation of CTLs depends on the presence of certain
cytokines, among others, IL-2 and IFN-y,”.” which are secreted by activated T-helper type 1 cells (Thl cells). Activation of these T-helper cells depends on antigen presentation
in the context of MHC class I1 (MHC-11) molecules by antigen presenting cells.23Occasionally, other nonlymphoid cells
like neoplastic epithelial cells have been described to express
MHC-11, which might assist in the onset of the cellular immune response.”
The aim of the study was to get insight in the cellular
immune response against H-RS cells by analyzing the cellular composition around H-RS cells in relation to the expression of MHC-I, MHC-11, and TAP- 1 on H-RS cells in EBV’
and EBV- cases of HD.
HE DIAGNOSIS OF Hodgkin’s disease (HD) is based
on the presence of typical Reed-Stemberg cells (RS
cells) and their mononuclear variants, Hodgkin cells (H
cells), in an appropriate cellular environment of “reactive”
cells.’ The presence of Epstein-Barr virus (EBV) in the neoplastic cells has been shown in approximately 40% of all
cases of HD, depending on histologic type.* In these EBVharboring cells expression of a restricted set of viral latent
genes was detected, ie, EBV nuclear antigen 1 (EBNAI)
and three latent membrane proteins (LMPI , LMP2A, and
LMP2B).3-6Despite the fact that LMPl and LMP2 contain
epitopes recognized by cytotoxic T lymphocytes (CTLs)
when presented in the context of appropriate major histocompatibility complex class I (MHC-I) molecule^,^^' HD patients apparently cannot raise an effective immune response
against these EBV-harboring neoplastic cells.
There is some evidence that patients with HD exhibit a
defect in cell-mediated immunity.’,’”These defects include
an impaired response to T-cell mitogens” and a decreased
capacity of T cells to respond in an autologous mixed lymphocyte response.‘*Moreover, in vitro studies showed a decreased synthesis of interleukin-2 (IL-2) and interferon-?
(IFN-y) (both cytokines with a T-cell-stimulating effect) in
untreated patients with HD.” However, the contribution of
this immune defect to the pathogenesis of HD is still obscure.
The major effector cells in cellular cytotoxicity are CTLs
From the Department of Pathology, Free University Hospital Amsterdam, and the Department of Pathology, University Hospital
Leiden, Leiden, The Netherlands.
Submitted August 24, 1995; accepted December 11, 1995.
Supported by u grant from the Dutch Cancer Society (VU 94749).
Address reprint requests to J.J. Oudejuns, MD, Department oj
Pathology, Free University Hospital, PO Box 7057, 1007 MB Amsterdam, The Netherlands.
The publication costs of this article were dejrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8709-0044$3.00/0
3844
Blood, Vol 87,No 9 (May 1). 1996:pp 3844-3851
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
3845
MHC CLASS I EXPRESSION ON RS CELLS
The results show that, compared with EBV- cases of HD,
H-RS cells in EBV+ cases express high levels of MHC-I.
Although these high MHC-I expression levels were accompanied by significantly higher numbers of activated CTLs
(ie, CD8 and granzyme B+ cells), these cells were only
sporadically detected in the close vicinity of the H-RS cells.
These data suggest that the failure of the immune system to
eradicate EBV-harboring H-RS cells is due to local inhibition of the function of activated CTLs by H-RS cells or by
reactive cells in the vicinity of H-RS cells.
MATERIALS AND METHODS
Table 2. MoAbs Used in This Study
Antibody
Antigen
Titer
P/Fx
HCA2
HCIO
B2M
W6/32
LN3
HLA-A
HLA-B, C
&microglobulin
MHC-I complex
TAP-I
TAP-I
CD3
CD3
1:400
1:50
P/F
P/F
OPD4
CD45R05
1:25
P/F
HLA-DR
1:500
1:1,000
1:40/1:100
1:25
1:25§
P/F
P/F
P/F
F
P/F
Pretreatment1
10 min 95°C TUFS
-
10 min 100°C lead
thiocyanate
10 min 95°C TUF
10 min 100°C
citrate
10 min 100°C
water, trypsin
10 min 100°C
citrate
10 min 100°C
citrate
CD8
CD8
1:511
P/F
Tissues. Patients with HD were selected from the files of the
Department of Pathology of the Free University Hospital Amsterdam
GrB7
Granzyme B
1:50
P/F
(Amsterdam, The Netherlands; N = 34) and the Department of
Pathology, University Hospital Leiden (Leiden, The Netherlands; N
1:lOO
LMP-I
P/F
= 29). Cases were classified according to the Rye clas~ification.~~ SI2
1:lOO
CSI-4
LMP-1
P/F
Staging of the patients was done according to the Ann Arbor classificationZ6Patient and tumor characteristics are summarized in Table
* P, paraffin sections; F, frozen sections.
1. Representative tissue specimens were fixed in buffered formaldet Only for formalin-fixed, paraffin-embedded tissue sections.
hyde or in a sublimate formaldehyde mixture. Paraffin-embedded
*Target Unmasking Fluid, Kreatech, Amsterdam, The Netherlands.
tissues were cut to 5-pm thick sections and mounted on 3-amino5 Incubations were performed overnight.
propyl-triethoxy-silane (APES; Sigma, St Louis, M0)-coated slides
11 Strains mainly CD4’ cells, is not reactive with monocytes.
for RNA in situ hybridization (RISH) and on poly-L-lysine-coated
slides for immunohistochemical staining. When available, frozen
tissue sections, fixed in pure acetone for 15 minutes, also were
endogenous peroxidase was blocked, and sections were pretreated
investigated. In total, frozen tissue sections of 9 EBV’ and 9 EBVaccording to the appropriate protocol for the different antibodies.
cases could be examined. Histologic features and EBV status have
Frozen sections were not pretreated, but transferred after acetone
partly been described elsewhere.”
fixation to phosphate-buffered saline (PBS). Both paraffin and frozen
Detection of the presence of EBV in H-RScells. The presence
sections were subsequently washed, preincubated with normal seof EBV in H-RS cells was determined by RISH using the abundantly
rum, and incubated with the primary antibody. To visualize the
transcribed noncoding EBV small RNAs (EBERI and 2) as has been
antibodies, a routine ABC immunoperoxidase method was used. The
described previouslyz7and by the detection of LMPl using two sets
following antibodies were used: MoAb HCA2, reactive with HLAof MoAbs (CSI-4: DAKO, Glostrup, Denmark and S12: Organon
A locus productsz9; MoAb HC 10, preferentially recognizing HLATeknika, Oss, The Netherlands).”
B/C locus products2’; polyclonal anti-&microglobulin B2M, recImmunohistochemistry. Immunohistochemical analysis for MHC
ognizing free and complexed @,-microglobulin (DAKO); MoAb
expression, phenotypic lymphocyte markers, and granzyme B exLN3 (Biotest Seralc, Brussels, Belgium), specific for HLA-DR; W6/
pression was performed as described p r e v i o ~ s l y ?Briefly,
~
formalin32 (Seralab, Sussex, UK), recognizing HLA-A, -B, and -C locus
fixed, paraffin-embedded sections were deparaffinized with xylene,
products, complexed to &microglobulin; polyclonal antihuman
Table 1. Patient and Tumor Characteristics
EBV Status
Negative
(n = 381
Age
(range)
Sex M/F
Histology
NS
MC
LP
LD
Unclassifiable
Stage of presentation
Stage I
Stage II
Stage 111
Staae IV
36, 7
( 14-73)
19/19
30
5
2
1
0
Positive
In = 25)
38, 9
(10-78)
17/8
16
6
1
0
2
6
27
15
1
4
2
7
1
Abbreviations: NS, nodular sclerosing; MC, mixed cellularity; LP,
lymphocyte predominant; LD, lymphocyte depleted.
TAP- 1 Iy; polyclonal anti-CD3 (DAKO); because no CD4-specific
MoAbs are available for paraffin-embedded material, the OPD4
MoAb (DAKO) was used. This MoAb preferentially detects CD4+
T helper cells, but was officially assigned to the CD45RO cluster‘o.”;
MoAb anti-CD8 (a generous gift from Dr D.Y. Mason, Oxford,
UK); and MoAb GrB7, raised against recombinant granzyme B protein and specific for human granzyme B.14 MoAb GrB7 reacts with
isolated granzyme B from activated cytotoxic lymphocytes on immunob10t.I~In addition, GrB7 detects granzyme-B-expressing cells in
routinely formalin-fixed paraffin-embedded tissue sections.3z Pretreatment and incubation conditions are summarized in Table 2.
Phenotypical analysis of grunzyme-B-expressing cells. In five
cases, double stainings were performed for either granzyme B and
CD3, granzyme B and OPD4, or granzyme B and CD8. After boiling
in a citrate buffer for 10 minutes, sections were incubated with GrB7,
followed by incubation with biotin-labeled goat-antimouse IgG2a
(Southem Biotechnology Associates Inc, Birmingham, AL). Subsequently, sections were incubated with peroxidase-conjugated streptavidin. The peroxidase was visualized by incubation for 10 minutes
in 0.2 mg/mL DAB, 0.002% H202,0.07% NiCll in 50 mmol/L TrisHCI, pH 7.6. After blocking the remaining peroxidase activity with
0.3% H202-methanol for 15 minutes, sections were incubated with
either CD3-, OPD4-, or CD8-specific antibodies. Subsequently, the
sections were incubated with either biotin-labeled swine-antirabbit
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
3846
OUDEJANS ET AL
(DAKO) for CD3 or biotin-labeled goat-antimouse-IgG1 (DAKO)
for OPD4 or CD8, followed by incubation with the streptavidinbiotin-horseradish peroxidase complex. Visualization was done with
diaminobenzidene (DAB)/H202, resulting in a clear brown staining
signal. Subsequently, silver enhancement of the DAB-nickel precipitate was performed as described previo~sly,’~
resulting in a black
staining signal for granzyme-B expression. Negative controls included simultaneously processed sections with omission of the GrB7
and CD3-, OPD4- or CD8-specific antibodies, respectively.
Interpretation of immunohistochemical stainings. Evaluation of
the staining levels was performed as described el~ewhere’~:only
those tissue sections in which the surrounding reactive cells showed
homogeneous staining were analyzed. The staining intensities observed in surrounding lymphocytes and histiocytes with the different
MHC markers was considered to be 2+ (moderate staining intensity).
If the staining intensity was weaker, the staining was scored as 1+;
if stronger it was scored as 3 + . TAP- 1 staining was scored as negative or positive. Using this protocol, the stainings were analyzed
independently by three observers. In case of disagreement the staining results were reanalyzed by the observers until consensus was
reached. Quantification of the relative numbers of OPD4, CD8, and
GrB7+ lymphocytes was performed using a commercially available
interactive video-overlay based measuring system (Q-PRODIT;
Leica, Cambridge, UK). The microscopal image is recorded by the
video camera and displayed on the computer screen. Selected corresponding areas in the sections stained with the respective antibodies
were demarcated. In these areas 10 to 20 fields of vision were
systematically randomly selected using an automatic scanning stage
controlled by Q-PRODIT. In these fields 200 to 400 lymphocytes
were scored positive or negative for OPD4, CD8, and GrB7 using
a point sampling method with a Weibel test grid.35A 40X objective
was used. This gives a final magnification of 1,200X on the computer
screen, and appeared the most suitable for this purpose. The number
of OPD4, CD8, and granzyme B+ cells was expressed as percentage
of all lymphocytes as judged by morphology.
To see whether the percentage of granzyme-B-expressing cells
was merely a reflection of the number of neoplastic cells present,
the number of CD30’ H-RS cells was estimated on several representative areas of the tumor. Cases were divided into four categories;
less than 1 neoplastic cell per medium power field (mpf), 1 to 3 per
mpf, 3 to 10 per mpf, and more than 10 per mpf.
Statistical analysis. Categorical data were analyzed using the
Pearson x2 test or the Fisher’s exact test when appropriate. The
Mann-Whitney U-test was used to compare group means and the
Spearman test was used to test correlations between the different
variables, considering P < .05 as significant. All analyses were
performed using the SPSS statistical software (SPSS Inc, Chicago,
ILl
RESULTS
MHC-I, MHC-II, and TAP-I expression in H-RS cells.
Frozen as well as formalin-fixed, paraffin-embedded tissue
sections were available from 18 cases of HD (9 EBV+ and
9 EBV- cases). In these cases, no major discrepancies in
immunohistochemical staining intensities between frozen
and paraffin-embedded tissue sections were observed. Because the availability of frozen material was limited and
interpretation of the immunohistochemical staining on frozen tissue sections was relatively difficult, we choose to
expand the study on paraffin-embedded tissue sections only.
The results of MHC-I, MHC-11, and TAP-1 expression in
H-RS cells in relation to EBV status of these HD cases are
Table 3. MHC-I Expression on H-RS Cells
in EBV- and EBV’ Cases of HD
EBV Status
HLA-A
0
1
2
3
ni
HLA-BIC
0
1
2
3
ni
&Microglobulin
0
Negative
Positive
n
n = 25
0
1
6
16
2
n = 25
0
2
12
11
0
n = 25
0
1
14
10
0
n = 9
0
0
9
0
n = 25
1
9
10
5
0
n = 19
17
0
2
n
n
1
2
3
ni
W6l32
0
1
2
3
HLA-DR
0
1
2
3
ni
TAP-1
Positive
Negative
ni
n
n
n
38
12
13
6
1
6
= 38
21
12
2
3
0
= 38
6
16
15
1
0
= 9
1
1
7
0
= 38
6
11
17
3
1
= 29
28
1
0
=
P Value‘
All cases P < ,001
NS P < ,001
MC P = .03
All cases P < ,001
NS P < .001
M C P = .01
All cases P < ,001
NS P < .001
MC P = .04
ns
ns
ns
Abbreviations: ni, not interpretable; ns, not significant.
* As determined by Pearson x 2 test, by Fisher‘s exact test.
summarized in Table 3 . In all tissue sections the reactive
nonneoplastic cells served as a positive control for staining
with the MHC-I-specific antibodies. Expression of MHC-I
heavy chains was predominantly observed on the cell membrane. This is in agreement with a previous report showing
intense membranous reactivity of both the HCA2 and HClO
antibodies by immunoelectron micro~copy.~~
In most EBV+ cases, even at low magnification, the atypical cells were easily recognized because the intensity of
the immunohistochemical staining was stronger than in the
reactive cells (see Fig 1A and B). This in contrast to the
EBV- group where the staining intensity of H-RS cells was
frequently weaker than in the surrounding reactive cells (Fig
1E and F). Cases in which reactive cells were not clearly
positive using the different anti-MHC and TAP-1 antibodies
were considered as not interpretable.
In the 25 EBV’ cases strong staining of the H-RS cells
(staining intensity 2 or 3 ) was observed in 22, 23, and 24
cases for HLA-A, HLA-B/C, and &macroglobulin, respectively, whereas in the 35 EBV- cases strong staining was
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
MHC CLASS I EXPRESSION ON RS CELLS
3847
.
Fig 1. Three cases of HD
showing differences in MHC-Iexpression. Hematoxilin counterstaining. Original magnificetion
~600.
EBV+ HD expressing high
levels of HLA-A (A) and HLA-B/C
IB, staining intensity scored as
3+1. EBV+HD expressing normal
levels of HLA-A IC) and HLA-B/C
ID, staining intensity scored as
2+1. EBV-HD expressing no detectable levels of HLA-A (El and
HLA-B/C IF, staining intensity
scored as 01.
I
Fig 2. TAP-1 expression in HD. Clear cytoplasmic staining is observed in the H-RS
cells end in most surrounding lymphocytes.
Fig 3. Phenotype of granzyme-B-expressing cells. Black staining indicates granzyme-B expression, brown staining indicates either OPD4
or CD8 expression. Hematoxilin counterstaining. Original magnification x 900. [AI EBV+ HD case, showing many granzyme B+/CD8+cells. (B)
EBV- HD case, showing many OPD4+ cells, granzyme B+ cells are OPD4-.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
3848
only observed in 7,5, and 16 cases, respectively ( P < ,001).
These differences were still significant when nodular sclerosing (NS) and mixed cellularity (MC) HD were analyzed
separately (see Table 3), excluding the possibility that differences in MHC-I expression are primarily related to histologic
features. In contrast, no differences between EBV+ and
EBV- cases were observed for MHC-I1 expression using
the LN3 antibody recognizing HLA-DR. In both EBV+ and
negative HD cases, strong expression of HLA-DR in H-RS
cells was detected in approximately 50% of cases.
TAP-1 expression was detected in the cytoplasm of H-RS
cells in nearly all cases of HD tested (Fig 2). In only 1
EBV- case no TAP- 1 positive H-RS cells could be detected,
whereas the surrounding reactive cells showed clear positive
signals. This TAP- 1 case was also negative for HLA-A and
HLA-B/C, whereas &macroglobulin was clearly detectable.
Detection of MHC-I in H-RS cells using the W6/32 antibody. In cases where frozen material was available (9
EBV' and 9 EBV- cases), expression of MHC-I molecules
was also studied using the W6/32 antibody and compared
to the staining intensities obtained with HLA-A-, HLA-B/
C-, or P2-macroglobulin-specific antibodies. In all 9 EBV'
HD cases comparable strong staining intensities on H-RS
cells were observed with both the W6/32 antibody and the
other MHC-I-specific antibodies. In the EBV- cases H-RS
cells were sometimes stained more intensely using W6/32
compared with the other MHC-I-specific antibodies. In 2
of 9 EB- cases low expression of all MHC-I-specific markers was observed, including W6/32. In the remaining 7 cases
with clear W6/32+ signals, 3 cases showed clear staining
signals for HLA-A using the HCA2 MoAb, 4 cases for HLAB/C using the HClO MoAb, and 5 cases for ,&-microglobulin.
In situ detection of (activated) cytotoxic cells. In all 45
tested cases both OPD4 and CD8+ lymphocytes were observed. Identical areas on consecutive tissue sections were
selected for quantification. In the EBV' cases of HD the
OPD4/CD8 ratio was significantly lower ( 1 3) than in the
EBV- cases (2.9, P = ,004, see Table 4). The relatively low
OPD4/CD8 ratio in EBV+ cases was mainly caused by a
significantly higher mean percentage of CD8' cells (34% L'
21%, P = .002). Identical levels of significance were obtained when statistical analysis was restricted to NS and MC
HD cases. In addition, a significant correlation was observed
between the number of CD8' cells and expression of HLAA ( r = .31, P = .04), HLA-B/C ( r = .45, P = .002), and
&microglobulin ( r = .35, P = .02) on H-RS cells, irrespective of EBV status.
In 57 of 61 cases tested, granzyme B' small reactive
lymphocytes were found. As expected, a granular staining
pattern was observed in these cells using the GrB7 antibody
(Fig 3). The percentage of lymphocytes expressing granzyme
B ranged from less than 10% in the majority of cases to
more than 20% in 5 cases. However, granzyme B+ cells
were only sporadically detected in the close vicinity of the
H-RS cells. The mean percentage of granzyme B+ cells was
significantly higher in the EBV' cases (1 1.4%) compared
with the EBV- cases (6.5%, P = .02, see Table 4). When
statistical analysis was restricted to NS and MC and HD
OUDEJANS ET AL
Table 4. Quantification of Reactive Lymphocytes
in EVB- and EBV' Cases of HD
EBV Status
Percentage of OPD4'
lymphocytes
Mean
(SD)
Percentage of CD8lymphocytes
Mean
(SD)
OPD4lCD8 ratio
Mean
(SD)
Percentage of granzyme B '
lymphocytes
Mean
(SD)
Negative
Positive
n = 27
48%
(18)
n = 18
38% Ail cases P = ,057
(11) NS and MC P = .060
n = 27
21%
(14)
n = 27
2.9
(2.2)
n = 18
34%
(15)
n = 18
1.3
(0.7)
n=33
6.5%
(5.8%)
n=24
11.4% All cases P = .015
(8.7%) NS and MC P = .04
PValue'
All cases P = ,002
NS and MC P = ,002
All cases P = ,004
NS and MC P = .005
* A s determined by Mann-Whitney U-test.
cases, the mean percentages were 11.1 and 6.3 for EBV'
and EBV- cases, respectively ( P = .04). Also irrespective
of EBV status, the number of GrB7' lymphocytes correlated
well with expression levels of HLA-A ( r = .45, P = ,003)
and HLA-B/C ( r = .41, P = .006), whereas no significant
correlation was found with P2 microglobulin ( r = .14, P =
.I@. The estimated number of H-RS cells per medium power
field was not found to correlate with the number of activated
CTLs ( r = .13, P = .26), suggesting that relatively high
numbers of activated CTLs cannot simply be explained by
the presence of high numbers of neoplastic cells only.
Phenotype qf grunzyme-B-expressing cells. GranzymeB expression has been detected in activated CTLs and in
NK cells." Double-staining experiments were performed to
determine whether granzyme-B-expressing cells were either
T-helper cells (CD3+/OPD4+),CTLs (CD3'/CD8+), or NK
cells (CD3 -). The results showed that the large majority of
granzyme-B-expressing cells were CD3'/CD8+/OPD4(Fig 3A and B), with sporadic GrB7+/OPD4' and GrB7'/
CD3 cells. This indicates that the majority of granzymeB-expressing cells were activated CTLs. This was further
substantiated by the strong correlation between the number
of GrB7+ cells and the number of CD8+ cells ( r = .48, P
,001).
Clinical features. No major differences were found in
clinical presentation between EBV+ and E B V cases (Table
1). In both groups, patients usually presented with stage I
or stage I1 disease at time of diagnosis with tumor localizations in one or several lymph nodes in the neck region.
=
DISCUSSION
We have shown that H-RS cells in EBV' HD cases express significantly higher levels of MHC-I compared with
EBV- cases. High MHC-I expression levels were accompanied by higher numbers of activated CTLs. No such differences were observed in MHC-I1 expression.
In a significant number of cases downregulation of MHC-
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
MHC CLASS I EXPRESSION ON RS CELLS
3849
cells were rarely present in the close vicinity of the H-RS
I expression on the surface of H-RS cells was found. MHC-I
downregulation was also observed by Poppema and V i s ~ e r . ~ ~cells suggests that activated CTLs are either inhibited in their
cytotoxic function by H-RS cells or by reactive cells in
However, in our study, these decreased levels of MHC-I
the vicinity of H-RS cells or fail to recognize the antigens
expression were mainly observed in the EBV- cases of HD,
presented by MHC-I molecules on the surface of the H-RS
whereas Poppema and Visser reported a lack of MHC-I excells.
pression levels in nearly all cases. These discrepancies might
Interestingly, similar conclusions were drawn in a recent
partly be explained by the fact that, except for W6/32, differarticle by Frisan et al.42 In their study, tumor-infiltrating
ent antibodies were used. That, in contrast to Poppema and
lymphocytes isolated from patients with EBV' HD lacked
Visser, our study was performed not on frozen material but
EBV-specific cytotoxicity, whereas EBV-specific cytotoxicprimarily on paraffin-embedded material cannot explain the
ity was detected in CTL precursors derived from the blood
discrepancies, because in a small group (n = 18) we found
of one of these patients. In line with our data, this lack of
comparable results on both frozen and paraffin-embedded
EBV-specific cytotoxicity was not associated with decreased
tissue sections.
numbers of CTLs present in the biopsy specimens of these
The presence of HD cases harboring H-RS cells with low
EBV' HD patients.
levels of MHC-I expression can well be explained by a
Several mechanisms might explain this ineffective CTL
positive selection for these cells in immunocompetent indiresponse. First, alterations in EBV-encoded proteins may
viduals, as has been suggested before.3hHowever, the mechaffect antigen presentation or modify immunogenic epitopes
anism leading to this partial or complete loss of MHC-I
that are relevant for T-cell recognition. It has been shown
expression has yet to be unravelled. Unlike cervical carcinothat in a murine model system, tumor cells expressing a
mas, where downregulation of MHC-I expression was
mutated form of LMPl, isolated from a nasopharyngeal carstrongly associated with absence of the TAP-1 transporter,"
cinoma biopsy sample, were not recognized by cytotoxic T
no such downregulation of TAP-1 was observed in HD cases
~ e l l s . 4In~ a proportion of cases of HD, the LMPl gene was
with low expression levels of MHC-I. In a recent report, a
dissociation of MHC-I and TAP expression was also found
found to harbor similar mutations.44
in certain cell lines.37Although expressing the appropriate
Another mechanism could be that EBV-encoded proteins
MHC-I molecule, endogenous processing of specific epiare recognized by activated CTLs, but that the function of
topes was impaired in these cell lines, whereas no apparent
these CTLs is inhibited by certain cytokines secreted by the
abnormalities were observed in the function of TAP-I and
H-RS cells or by reactive cells in the vicinity of H-RS cells.
Among cytokines that inhibit cell-mediated cytotoxicity are
TAP-2.'7
Our data indicate that the level of MHC-I expression on
IL- 1045-48
and transforming growth factor-@(TGF-p),49.50
of
H-RS cells and the number of activated cytotoxic cells preswhich TGF-@expression has been detected in an HD-derived
ent at the tumor site is related to the presence of EBV in Hcell line.51Interestingly, LMPl has been shown to upregulate
RS cells. If the CTLs are directed against the H-RS cells,
IL-10s2 and one of many EBV genes (BCRFl) encodes a
the differences in numbers of activated CTLs might be exprotein that is highly homologous to IL-10.s3 Recently, explained by the presence of "non-self," ie, viral antigens in
pression of BCRFl as well as IL-10 has been reported in
EBV' HD and the absence of such viral antigens in EBVEBV' cases of HD.54 Thus, expression of either TGF-P or
cases. Putative targets for a CTL response in EBV' HD
viral or human IL- 10 in H-RS cells could explain the failure
are LMPl and LMP2?.9.38..39
However, the possibility that
of the activated CTLs to eradicate EBV-harboring H-RS
another, yet unknown virus is present in H-RS cells of EBVcells.
In conclusion, we have shown that H-RS cells in EBV'
HD cannot be excluded.
HD cases express relatively high levels of MHC-I, which
Apart from the ability of LMPl to function as target for
are accompanied by increased numbers of CD8 and granCTLs, the results of several reports have shown that LMPl
zyme B+ cells. Because activated CTLs were only sporadiis able to increase the overall immunogenicity of EBV-harcally detected in the close vicinity of the H-RS cells, we
boring cells through upregulation of molecules in the antigen
suggest that local factors (ie, cytokines) expressed by H-RS
presentation pathway.@"' LMPl was shown to be able to
cells or reactive cells in the vicinity of H-RS cells inhibit
upregulate expression of the TAP proteins and of MHC-I
the function of activated CTLs, resulting in an inefficient
molecules, which was accompanied by restoration of CTL
immune response against H-RS cells.
recognition of endogenously processed viral antigen^.^'
Still, different from the cases with low levels of MHC-I
expression in H-RS cells, the failure of the immune system
ACKNOWLEDGMENT
to eradicate the neoplastic cells in cases where H-RS cells
The authors thank Gemt Meijer for his excellent assistance in
display normal or high levels of MHC-I expression and harstatistical analysis of the data, Prof H.L. Ploegh for critically reading
bor relatively high numbers of activated CTLs remains difthe manuscript, and Dr J.M. Middeldorp for providing the SI2 antificult to explain. Activation of cytotoxic cells depends on
body.
specific MHC-I-restricted antigen recognition and on the
presence of costimulatory cytokines secreted by Th- 1 cells.
REFERENCES
Because activated CTLs were present, inhibition of the func1. Kadin ME: Hodgkin's disease: Immunobiology and pathogenetion of cytotoxic cells probably occurs after these cells have
sis, in Knowles DM (ed): Neoplastic Hematopathology. Baltimore,
been activated. However, the observation that granzyme B'
MD, Williams and Wilkins, 1992, p 535
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
3850
2. Herbst H, Niedobitek G: Epstein-Barr virus in Hodgkin’s disease. Epstein-Barr Virus Report 1:31, 1994
3. Deacon EM, Pallesen G , Nieboditek G, Crocker J, Brooks L,
Rickinson AB, Young LS: Epstein-Barr virus and Hodgkin’s disease:
Transcriptional analysis of virus latency in the malignant cells. J
Exp Med 177:339, 1993
4. Joske DJL, Emery-Goodman A, Bachmann E, Bachmann F,
Odermatt B, Knecht H: Epstein-Barr virus burden in Hodgkin’s
disease is related to membrane protein gene expression but nor to
active viral replication. Blood 80:2610, 1992
5. Herbst H, Dallenbach F, Hummel M, Niedobitek G, Pileri S,
Mueller-Lantzsch N, Stein H: Epstein-Barr virus latent membrane
protein expression in Hodgkin and Reed-Stemberg cells. Proc Natl
Acad Sci USA 88:4766, 1991
6. Grasser FA, Murray PG, Kremmer E, Klein K, Remberger K,
Feiden W, Reynolds G, Niedobitek G, Young LS, Mueller-Lantzsch
N: Monoclonal antibodies directed against the Epstein-Barr virusencoded nuclear antigen 1 (EBNA 1): Immunohistologic detection
of EBNAI in the malignant cells of Hodgkin’s disease. Blood
84:3792, 1994
7. Masucci M, Emberg I: Epstein-Barr virus: Adaptation to a life
within the immune system. Trends Microbiol 2:125, 1994
8. 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
9. Murray R, Kurilla M, Brooks J, Thomas W, Rowe M, Kieff
E, Rickinson A: 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
176157, 1992
10. Slivnick DJ, Ellis TM, Nawrocki JF, Fisher RI: The impact
of Hodgkin’s disease on the immune system. Semin Oncol 17:673,
1990
11. Levy RA, Kaplan HS: Impaired lymphocyte function in untreated Hodgkm’s disease. N Engl J Med 290: 181, 1974
12. Engleman EG, Benike CJ, Hoppe RT, Kaplan HS, Berberich
FR: Autologous mixed lymphocyte reaction in patients with Hodgkin’s disease. Evidence for a T cell defect. J Clin Invest 66:149,
1980
13. Ford RJ, Tsao J, Kouttab NM, Sahasrabuddhe CG, Metha
SR: Association of an interleukin abnormality with the T-cell defect
in Hodgkin’s disease. Blood 64:386, 1984
14. Kummer JA, Kamp AM, van Katwijk M, Brakenhof JP, Radosevic K, van Leeuwen AM, Borst J, Verwey CL, Hack C E Production and characterization of monoclonal antibodies raised against
recombinant human granzymes A and B and showing cross reactions
with the natural proteins. J Immunol Methods 163:77, 1993
15. Shiver JW,
Lishan SU, Henkart PA: Cytotoxicity with target
DNA breakdown by rat basophilic leukaemia cells expressing both
cytolysin and granzyme A. Cell 71:315, 1992
Wesselschmidt RL, Shresta S, Russell JH, Ley TJ:
16. Heusel JW,
Cytotoxic lymphocytes require granzyme B for the rapid induction of
DNA fragmentation and apoptosis in allogenic target cells. Cell
76977, 1994
17. Garcia-Sanz JA, Macdonald HR, Jenne DE, Tschopp J, Nabholz M: Cell specificity of granzyme gene expression. J Immunol
145:3111, 1990
18. Monaco JJ: A molecular model of MHC class-I-restricted
antigen presentation. Immunol Today 13:173, 1992
19. Cromme FV,Airey J, Heemels MT, Ploegh HL, Keating PJ,
Stem PL, Meijer CJLM, Walboomers JMM: Loss of transporter
protein, encoded by the TAP-1 gene, is highly correlated with loss
of HLA expression in cervical carcinomas. J Exp Med 179:335,
I994
OUDEJANS ET AL
20. Trowsdale J, Hanson I, Mockridge I, Beck S, Townsend A,
Kelly A: Sequences encoded in the class I1 region of the MHC
related to the ABC superfamily of transporters. Nature 348:741,
1990
21. Paul WE, Seder RA: Lymphocyte responses and cytokines.
Cell 76:24 1, 1994
22. Biron CA: Cytokmes in the generation of immune responses
to, and resolution of, virus infection. Curr Opin Immunol 6:530,
1994
23. Unanue, ER: Cellular studies on antigen presentation by class
I1 MHC molecules. Cum Opin Immunol 4:63, 1992
24. Glew SS, Duggan-Keen M, Cabrera T, Stem PL: HLA class
I1 antigen expression in human papillomavirus-associated cervical
cancer. Cancer Res 52:4009, 1992
25. Lukes RJ, Craver LF, Hall TC, Rappaport H, Ruben P: Report
of the nomenclature committee. Cancer Res 26:1311, 1966
26. Carbonne PP, Kaplan HS, Musschof K, Smithers DW: Report
of tbe committee on Hodgkin’s disease staging classification. Cancer
Res 31:1860, 1971
27. Jiwa NM, Kanavaros P, van der Valk P, Walboomers JMM,
Horstman A, Vos W, Mullink H, Meijer CJLM: Expression of c-myc
and bcl-2 oncogene products in Reed Stemberg cells independent of
presence for EBV. J Clin Pathol 4621 I , 1993
28. Jiwa NM, Oudejans JJ, Dukers DF, Vos W, Horstman A, van
der Valk P, Middeldorp JM, Kluin PhM, Walboomers JMM, Meijer
CJLM: Immunohistochemical demonstration of different latent
membrane protein-1 epitopes of Epstein-Barr virus in lymphoproliferative diseases. J Clin Pathol 48:438, 1995
29. Stam NJ, Vroom TM, Peters PJ, Pastoors EB, Ploegh HL:
HLA-A and HLA-B-specific monoclonal antibodies reactive with
free heavy chains in Westem blots, in formalin fixed, paraffin embedded tissue sections and in cryo-immuno-electron microscopy. lnt
Immunol 2:113, 1990
30. Yoshino T, Mukuzono H, Aoki H, Takahashi K, Takeuchi T,
Kubonishi I, Ohtsuki Y, Motoi M, Akagi T: A novel monoclonal
antibody (OPD4) recognizing a helperhnducer T cell subset. Am J
Pathol 134:1339, 1989
31. Poppema S, Lai R, Visser L: Monoclonal antibody OPD4 is
reactive with CD45R0, but differs from UCHLl by the absence of
monocyte reactivity. Am J Pathol 139:725, 1991
32. Kummer JA, Kamp AM, Tadema TM, Vos W, Meijer CJLM,
Hack E Localization and identification of granzymes A and B expressing cells in normal human lymphoid tissue and peripheral blood.
Clin Exp Immunol 100:164, 1995
33. Merchentaler I, Stankovics J, Gallyas F: A highly sensitive
one-step method for silver intensification of the nickel-diaminobenzidine end product of peroxidase reaction. J Histochem Cytochem
37:1563, 1989
34. Jiwa NM, Oudejans JJ, Bai MC, van den Brule AJC, Horstman A, Vos W, van der Valk P, Kluin PhM, Walboomers JMM,
Meijer CJLM: Expression of bcl-2 protein and transcription of the
Epstein-Barr virus bcl-2 homologue BHRFl in Hodgkin’s disease:
Implications for different pathogenic mechanisms. Histopathology
26547, 1995
35. Fleege JC, van Diest PJ, Baak JPA: Computer assisted efficiency testing of different sampling methods for selective nuclear
graphic tablet morphometry. Lab Invest 63:270, 1990
36. Poppema S, Visser L: Absence of HLA class I expression by
Reed-Stemberg cells. Am J Pathol 145:37, 1994
37. Rowlan-Jones S, Powis SH, Sutton J, Mockridge 1, Gotch
FM,Murray N, Hill AB, Rosenberg WM,Trowsdale J, McMichael
AJ: An antigen processing polymorphism revealed by HLA-B8restricted cytotoxic T lymphocytes which does not correlate with
TAP gene polymorphism. Eur J Immunol 23:1999, 1993
38. Lee SP, Thomas WA, Murray RJ, Khanim F, Kaur S, Young
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
MHC CLASS I EXPRESSION ON RS CELLS
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
39. Thorley-Lawson DA, Israelson ES: Generation of specific
cytotoxic T cells with a fragment of the Epstein-Barr virus encoded
p63Aatent membrane protein. Proc Natl Acad Sci USA 84:5384,
1987
40. De Campos-Lima PO, Torsteinsdottir S, Cuoma L, Klein G,
Sulitzeana D, Masucci G: Antigen processing and presentation by
EBV carrying cell lines: Cell phenotypes dependence and influence
of the EBV encoded LMPl. Int J Cancer 53:856, 1993
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
42. Frisan T, Sjoberg J, Dolcetti R, Boiocchi M, De Re V, Carbone A, Brautbar C, Battat s, Biberfeld P, Eckman M, Ost A, Christensson B, Sundstrom C, Bjokholm 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
43. Trivedi P, Hu L, Chen F, Christensson B, Masucci M, Klein
G, Winberg G: The EBV encoded membrane protein LMPl from a
nasopharyngeal carcinoma is nonimmunogenic in a murine model
system, in contrast to a B-cell derived homologue. Eur J Cancer
30A:84, 1994
44. Knecht H, Bachmann E, Brousset P, Sandvej K, Nadal D,
Bachmann F, Odermatt BF, Delsol G, Pallesen G: Deletions within
the LMPl oncogene of Epstein-Barr virus are clustered in Hodgkin’s
disease and identical to those observed in nasopharyngeal carcinoma.
Blood 82:2937, 1993
45. Modlin RL, Nutman TB: Type 2 cytokines and negative immune regulation in human infections. Cum Opinion Immunol 5:511,
1993
385 1
46. Taga K, Tosato G: IL-10 inhibits human T-cell proliferation
and IL-2 production. J Immunol 148:1 143, 1992
47. Ding L, Shevach E M IL-10 inhibits mitogen induced Tcell proliferation by selectively inhibiting macrophage costimulatory
function. J Immunol 148:3133, 1992
48. 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- and allo-specific cytotoxic T
cells and downregulates HLA class I expression. J Exp Med
180:2371, 1994
49. Kerhl JH, Wakefield LM, Roberts AB, Jakowlew S, AlvarezMon M, Derynck R, Spom MB, Fauci AS: Production of transforming growth factor-beta by human T-lymphocytes and its potential role in the regulation of T-cell growth. J Exp Med 163:1037,
1986
50. Rook AH, Kerhl JH, Wakefield LM, Roberts AB, Spom MB,
Burlington DB, Lane HC, Fauci AS: Effects of transforming growth
factor B on the functions of natural killer cells: Depressed cytolytic
activity and blunting of interferon responsiveness. J Immunol
136:3916, 1986
51. Newcom SR, Kadin ME, Ansari AA, Diehl V: L-428 nodular sclerosing Hodgkin’s cell secretes a unique transforming
growth factor-beta active at physiologic pH. J Clin Invest 83:1915,
1988
52. Nakagomi H, Dolcetti R, Bejarano MT, Pisa P, Kiessling R,
Masucci MG: The Epstein-Barr virus latent membrane protein-I
(LMPI) induces interleukin- 10 production in Burkitt lymphoma
lines. Int J Cancer 57:240, 1994
53. Moore KW, Vieira P, Fiorentina DF, Trounstine ML, Khna
TA, Mosmann TR: Homology of cytokine synthesis inhibitory factor
(IL-10) to the Epstein-Barr virus gene BCRFl. Science 248:1230,
1990
54. Ohshima K, Suzumiya J, Akamatu M, Takeshita M, Kikuchi
M: Human and viral interleukin-10 in Hodgkin’s disease, and its
influence on CD4+ and CD8+ T lymphocytes. Int J Cancer 62:5,
1995
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1996 87: 3844-3851
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 -negative Hodgkin's disease
JJ Oudejans, NM Jiwa, JA Kummer, A Horstman, W Vos, JP Baak, PM Kluin, P van der Valk, JM
Walboomers and CJ Meijer
Updated information and services can be found at:
http://www.bloodjournal.org/content/87/9/3844.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.