CLINICAL RESEARCH

e-ISSN 1643-3750
© Med Sci Monit, 2016; 22: 2340-2346
DOI: 10.12659/MSM.896629

Received: 2015.11.11
Accepted: 2015.12.16 Classical Hodgkin Lymphoma with Positive
Published: 2016.07.05
Epstein-Barr Virus Status is Associated with
More FOXP3 Regulatory T Cells
Authors’ Contribution: ABDEF 1 Antonia Pavlovic 1 Department of Pathology, Forensic Medicine and Cytology, Clinical Hospital
Study Design  A ADEG 1 Merica Glavina Durdov Center, Split, Croatia
Data Collection  B 2 Department of Nuclear Medicine, Clinical Hospital Center, Split, Croatia
Analysis  C
Statistical CD 2 Vesna Capkun 3 Department of Hematology, Clinical Hospital, Split, Croatia

Data Interpretation  D BF 3 Jasminka Jakelic Pitesa 4 Department of Oncology, Clinical Hospital, Split, Croatia
Manuscript Preparation  E EF 4 Maja Bozic Sakic
Literature Search  F
Funds Collection  G

Corresponding Author: Antonia Pavlovic, e-mail: antonia.bendic@gmail.com

Source of support: The authors received financial support from the Ministry of Science, Education, and Sports of the Republic of Croatia

Background: Classical Hodgkin lymphoma (cHL) is characterized by sparse malignant Hodgkin and Reed-Sternberg cells dis-
persed in an inflammatory microenvironment. Immune evasion of malignant cells is partially due to the exis-
tence of a subpopulation of immunosuppressive regulatory T cells (Treg). The aim of this study was to analyze
T cell composition in cHL with special emphasis on Treg in regard to Epstein-Barr virus (EBV) status, subtype,
and patient age.
Material/Methods: The study included 102 patients with cHL diagnosed during a 12-year period. EBV status of cHL was assessed
immunohistochemically using antibodies directed to the EBV- encoded LMP1. To define T lymphocyte popula-
tions, slides were double-stained with FOXP3 for Treg, and CD4 or CD8 for T cells. In each case the number of
single- and/or double-positive cells was counted on an image analyzer in 10 high-power fields. Statistical anal-
ysis was performed and differences were considered significant at P<0.05.
Results: EBV-positive status of cHL was confirmed in 30 (29%) cases, mainly in patients older than 54 years and in
mixed cellularity subtype. In EBV-positive cHL, higher numbers of CD8+ cells were found. In cHL with positive
EBV status, more FOXP3+ Treg were found, as well as higher numbers of FOXP3+CD4+ Treg compared with
EBV-negative cHL. The number of CD4+ cells decreased with age. The frequency of FOXP3+CD8+ Treg was vari-
able, without a statistically significant association with age or EBV status.
Conclusions: EBV status has an impact on composition of T cell populations in the cHL microenvironment.

MeSH Keywords: Herpesvirus 4, Human • Hodgkin Disease • T-Lymphocytes, Regulatory

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FOXP3 Treg in classical Hodgkin lymphoma
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CLINICAL RESEARCH

Background immune privilege for tumor cells [21–25]. Unlike the majority
of solid tumors, in which high numbers of Treg are connect-
Hodgkin lymphoma (HL) is one of the most common lympho- ed to poor outcome, in HL it is associated with favorable out-
mas in the Western world: it accounts for 30% of all lympho- come [24,26–28]. Treg have been studied in HL in the context
mas and 15% of all malignancies in young adults [1,2]. On the of EBV status, but without identifying Treg subsets [26,29–31].
basis of difference in the morphology, phenotype of the lym- The aim of our study was to analyze T cell composition in cHL
phoma cells, and the composition of inflammatory infiltrate, HL with special emphasis on Treg and Treg subtypes in regard to
is divided into classical HL (cHL), subtyped into nodular scle- EBV status, cHL subtype, and patient age.
rosis (NS), mixed cellularity (MC), lymphocyte rich (LR), and
lymphocyte depleted (LD); and nodular lymphocyte predomi-
nant HL (NLPHL), which accounts for only 5% of HL [3,4]. In in- Material and Methods
dustrialized countries HL presents with a bimodal age distri-
bution, with a rise in incidence in young adults (20–34 years) This retrospective case-control study included patients di-
and in elderly people (55–75 years). In developing countries, agnosed with cHL in a 12-year period (1997–2009) at the
the first peak occurs earlier, in children and teenagers. Also, Department of Pathology, Forensic Medicine, and Cytology,
an inverse correlation between incidence of HL and socioeco- Clinical Hospital Center Split, Croatia. Paraffin blocks of tu-
nomic status was observed [5–7]. A prerequisite for the diag- mor tissue were retrieved from the Department’s archive, and
nosis of HL is malignant mononuclear Hodgkin cells and mul- were reviewed and classified according to WHO Classification
tinuclear Reed-Sternberg cells (HRS), which represent only of Tumors of Hematopoietic and Lymphoid Tissues, 2008. All
1–10% of the total tumor mass and reside in abundant in- samples were taken prior the initiation of treatment (cytostat-
flammatory infiltrate. The origin of HRS cell has long been an ic or irradiation). Inclusion criteria were: sufficient amount of
enigma due to the global loss of B cell phenotype and aber- tumor tissue in paraffin block, as well as all the necessary pa-
rant expression of markers characteristic of T cells or dendrit- tient data (age, sex, and HL subtype). All cases that did not
ic cells [3,4]. In 1994 Kuppers discovered that HRS are derived fulfill the criteria were excluded. A total of 102 cases were in-
from pre-apoptotic germinal center B cells that have acquired cluded. Approval for the study was obtained from the Hospital
disadvantageous “crippling” mutations, and in normal circum- Ethics Committee (code 2181-147-08-01/01-M.J.).
stances would have undergone apoptosis [8–10]. In less than
1% of cases, HRS is of T cell origin [11]. The pathogenesis of HL Paraffin-embedded tissue samples were cut into slices 3-μm-
is only partially understood and includes genetic and environ- thick, deparaffinized, and dehydrated. For antigen retrieval,
mental factors [2 6]. Genetic lesions frequently involve mem- slides were incubated for 20 min in TRIS/EDTA buffer, pH 9
bers of the NFkB family and the Jak-Stat signaling pathway, (Dako, Denmark). Endogenous peroxidase activity was blocked
pointing to their central role in the pathogenesis, but multiple by adding 200 μl of 3% H2O2. After rinsing in phosphate-buff-
signaling pathways exist and cooperate [3,4]. Epstein-Barr vi- ered saline (PBD), monoclonal antibodies were applied. EBV
rus (EBV) can be found in HRS in 40% of cHL in the Western status was assessed using LMP-1 mouse monoclonal anti-
world, and in 90–100% in developing countries, such as those body (clone CS1-4, Dako, Denmark), and results were inter-
in Africa and Latin America [5–7]. EBV-positive HL is more fre- preted based on brown membranous staining in HRS cells.
quent in children, older adult males, and mixed cellularity sub- For double-immunohistochemistry staining, the EnVisionTM
type of cHL [6]. Older individuals with EBV-positive HL have a G/2 Double stain System and Rabbit/Mouse DAB+Permanent
worse prognosis compared to young adults with EBV-positive Red, Dako, Denmark) were used according to the manufac-
HL, who have longer disease-free survival (DFS) [6,12]. In cHL, turer’s instructions.
EBV expresses a limited set of latent viral genes: EBNA1, LMP1,
LMP2A, EBER1, and EBER2 [13]. The tumor microenvironment Monoclonal mouse antibody against FOXP3 (236A/E7, Santa
of cHL is composed of mixtures of lymphocytes, plasma cells, Cruz Biotechnology, Inc.) was applied first (1:50), incubat-
macrophages, eosinophils, and mastocytes [14,15]. In the last ed overnight in a humidity-controlled chamber, and then the
decade, regulatory T cells (Treg) have been studied extensively staining process was finished in a Dako Autostainer using the
in non-tumor and tumor pathology [16]. Treg are developmen- standard manufacturer’s protocol. Consecutively, monoclonal
tally and functionally distinct T cell subpopulations engaged in antibody CD4 (clone 4 B12, Novocastra, UK, 1:100) or mono-
sustaining immunological self-tolerance and homeostasis [17]. clonal antibody CD8 (clone DK35, Dako, Denmark 1:100) was
FOXP3, a forkhead winged-helix transcriptional factor, is con- applied, incubated for 1 h, and the procedure was finished us-
sidered as a lineage-specific marker for Treg necessary for their ing the standard manufacturer’s protocol. DAB was used as a
development and function, as the majority of Treg are FOXP3- chromogen to identify FOXP3 and Permanent Red was used to
positive [17–22]. In the tumor microenvironment, Treg exert identify CD4 or CD8. Positive results were identified as brown
their suppressive activity on effector T cells (Teff), fostering nuclear staining of FOXP3 and red membranous staining for

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A B

C D

Figure 1. Double immunostaining for FOXP3 and CD4 and CD8 expression in classical Hodgkin lymphoma. FOXP3 expression is shown
as nuclear brown staining and CD4 or CD8 expression as membranous red staining. Double-positive cells show combined
nuclear brown and membranous red staining. (A) Numerous double-positive FOXP3/CD4 cells and single-positive CD4 cells.
(B) Rare double-positive FOXP3/CD4 cells and numerous single-positive CD4 cells. (C) Some double-positive FOXP3/CD8 cells
with single-positive CD8 cells. (D) No double-positive FOXP3/CD8 cell and some single-positive CD8 cells.

CD4 or CD8 (Figure 1). Slides were analyzed with an Olympus patients were younger than 26.5 years, 26.5% were 26.5–36
BX41 microscope at 1000×, photographed with an Olympus years, 25.5% were 36.1–54 years, and 23.5% were >54 years
digital camera, and analyzed with the Analysis program (Soft old. According to the expression of LMP1 in malignant cells,
Imaging System analysis program). The number of FOXP3 cells, EBV status was negative in 72 (71%) patients and was posi-
CD4, CD8, and dual-positive FOXP3+CD4+ and FOXP3+CD8+ tive in 30 (29%) patients. Subtypes were: 62% nodular sclero-
cells were counted in 10 random fields and expressed as to- sis (NS), 24.5% mixed cellularity (MC), and 13.5% lymphocyte-
tal cell number. rich (LR) and lymphocyte-depleted (LD) subtypes together. The
absolute numbers of FOXP3+, CD4+, CD8+, FOXP3+CD4+, and
For statistical analysis, the Mann-Whitney test, log-rank test, FOXP3+CD8+ cells in the microenvironment, counted in 10
and c2 test were used. Results were considered significant high-power magnification fields, are shown in Table 1.
at P<0.05.
There was a statistically significant correlation between EBV
status and patient age (c2=43.9; P<0.001). EBV-positive HL was
Results 13 times more frequent in patients older than 54 years than in
younger patients (p<0.001). Most of the EBV-positive HL were of
Of the 102 patients, 53 (52%) were males and 49 (48%) MC subtype (P=0.001). EBV-positive HL also had higher numbers
were females. Median age was 36 years (range, 5–87); 24.5% of FOXP3+ (c2=6.8; P=0.009) and CD8+ (c2=4.27; P=0.029) cells.

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Table 1. Distribution of T cell phenotypes in microenvironment Discussion

of classic Hodgkin lymphoma on 10 high power
magnification fields. Results of our study showed that EBV-positive cHL contains
generally higher number of FOXP3+Treg than EBV-negative
Phenotype Median (min-max) N cHL, and FOXP3+CD4+ Treg are the dominant Treg popula-
FOXP3+ 536 (17–2368) tion. In EBV-negative cHL, higher numbers of non-regulato-
ry CD4+ cells were found. We observed that the number of
CD4+ 2920 (708–5318)
CD4+ cells decreased with age at some point after age 26
FOXP3+CD4+ 414 (16–2355) years. Distribution of FOXP3+CD8+ Treg was so variable that
CD8+ 656 (33–2899) no clear associations were found. We found that 29% of cHL
were EBV-positive, and most of these patients were older than
FOXP3+CD8+ 4 (0–431)
54 years and had MC subtype, which is in concordance with
previous findings [1]. Our results show that our study popula-
Twice as many FOXP3+CD4+ cells were found in EBV-positive com- tion fit the “Western” population pattern, with approximately
pared with EBV-negative cases (c2=6.8 P=0.009). EBV-negative 30% EBV-positive cHL in immunocompetent patients. Previous
cases contained more CD4+ cells (c2=7.31; P=0.007) (Table 2). studies have shown that the cHL microenvironment is enriched
Correlation of cHL subtypes showed that NS and MC contained in Treg, and their high number could explain immune evasion
more FOXP3+ (p=0.014) and FOXP3+CD4+ cells compared to LD of HRS [29,30]. HRS cells express ligands CCL17, CCL22, and
and LR subtypes (c2=21.890, p=0.004) (data not shown). Galectin 1, which selectively attract CCR4 Treg to the cHL mi-
croenvironment [15,32]. HRS cells also express IL21, which up-
When correlating age groups with total number of all T cell regulates CC chemokine macrophage-inflammatory protein-3a
types, we found that patients younger than 26.5 years had high- and attracts FOXP3+ Treg [33]. Baumforth et al. found that in
er numbers of CD4+ cells compared to patients aged 26.5–36 EBV-positive cHL, EBNA1 upregulates expression of CCL20 in
years (c2=21.890, P=0.009). Distribution of FOXP3+CD8+ cells HRS cells, resulting in the increased migration of FOXP3+ cells
was so variable that no statistical differences were found. The to the microenvironment [31]. Although most authors agree
findings are summarized in Table 3. that the cHL microenvironment is enriched in Treg, no clear

Table 2. Correlation of EBV status with clinical and histological parameters in 102 cases of cHL.

LMP1– LMP1+
Parameter c2 OR (95%CI) P
N=72 (%) N=30 (%)

M 37 (51) 16 (53) 0.032 0.925 (0.394–2.17) 0.585

Sex
F 35 (49) 14 (47)

<54 68 (94) 10 (33) 43.90 3.4 (9.6–12.0) <0.001

Age
>54.1 4 (6) 20 (67)

NS 50 (70) 13 (43) 11.28 1.465 (0.829–2.59) 0.189

Subtype MC 11 (15) 14 (47)

LP and LD 11 (15) 3 (10)

<536.5 42 (58) 9 (30) 6.8 3.27 (1.3–8.1) 0.011

FOXP3+
>536.51 30 (41) 21 (70)

<3464 49 (68) 28 (93) 7.31 0.674 (0.451–1.006) 0.053

CD4+
>3464.1 23 (32) 2 (7)

<656.5 41 (57) 10 (33) 4.72 2.64 (1.09–6.45) 0.032

CD8+
>656.51 31 (43) 20 (67)

<414.5 42 (58) 9 (30) 6.8 3.27 (1.31–8.1) 0.011

FOX+CD4+
>414.51 30 (42) 21 (70)

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Table 3. Correlation of patient’s age with number of T cells.

Age (years)
T cells
N <26.5 26.5–36 36.1–54 >54 c2 P
parameter
N=25 (%) N=27 (%) N=26 (%) N=24 (%)
<227.75 7 (28) 3 (11) 10 (39) 5 (21) 9.650 0.380

227.75–536,5 8 (32) 6 (22) 7 (27) 5 (21)

FOXP3+
536.55–902 5 (20) 8 (30) 6 (23) 7 (29)

>902.1 5 (20) 10 (37) 3 (11) 7 (29)

<293 8 (32) 10 (37) 4 (15) 3 (12) 11.148 0.266

293.1–656.5 6 (24) 7 (26) 8 (31) 5 (21)

CD8+
656.5–1056.5 3 (12) 5 (18.5) 8 (31) 10 (42)

>1056.5 8 (32) 5 (18.5) 6 (23) 6 (25)

0.0001–4
FOX+CD8+
4001–30.75 1 (4) 1 (4) 1 (4) 1.117 0.773

>30.75 24 (96) 27 (100) 25 (96) 23 (96)

<2363 6 (24) 7 (26) 4 (15) 8 (33) 21.890 0.009

2363.1–2920.5 13 (48) 8 (31) 5 (21)

CD4+
2920.5–3464 9 (36) 4 (15) 6 (23) 7 (29)

>3464.1 10 (40) 3 (11) 8 (31) 4 (17)

<188.75 6 (24) 4 (15) 9 (35) 6 (25) 9.336 0.407

188.75–414.5 9 (36) 5 (18) 8 (31) 4 (17)

FOX+CD4+
414.5–811.75 5 (20) 8 (30) 6 (23) 7 (29)

>811.75 5 (20) 10 (37) 3 (11) 7 (29)

evidence of greater bias toward higher number of Treg in EBV- immunosuppressive cytokines, such as interleukin 10. This ob-
positive cHL has been found [26,27,29–31]. Results from a study servation suggests that differences in EBV-positive and EBV-
by Assis et al. were somewhat similar to ours. They performed negative cHL Treg pools could be even bigger and include new
qualitative and quantitative analysis of Treg in 130 cases of molecules and regulatory mechanisms [36]. Kyatsu et al. ana-
cHL in a Brazilian population and found that the presence of lyzed the T cell microenvironment in HIV-associated HL, which
EBV correlates with increased number of CD4+CD25+FOXP3+ is always an EBV-positive neoplasm. They found that the HIV-
Treg, but there was no association between Treg and clinical associated HL microenvironment contained significantly low-
characteristics [34]. Unlike Baumforth et al., we found higher er proportions of FOXP3+ Treg and higher proportions of CD8+
numbers of FOXP3+Treg and FOXP3+CD4+ cells in EBV-positive cells, compared to control HIV-negative HL patients. Also, in
cHL [31]. This may be because our study was performed with a the control group, EBV-positive HL was associated with more
larger number of patients. CD4+ lymphocytes are the dominant FOXP3+ cells and CD8+ cells than in EBV-negative HL, which
T cells in the cHL microenvironment [35]. We found increased is in agreement with our results [37]. Although the majority
numbers of CD4+ cells in EBV-negative cHL cases. According of CD8 cells are directed toward EBV antigens expressing la-
to Baumforth et al., this could partially be explained by the ab- tency III program, LMP1 and LMP2 can also act as immuno-
sence of EBNA1 effect on migration of Treg [31]. Morales et al. gens for CD8+ cells [38]. Kyatsu et al. reported that the abun-
recently reported that EBV is responsible for enrichment of the dant CD8+ cells present in HIV-associated HL probably are
HL microenvironment with regulatory Type 1 cells (Tr1) by in- not LMP-specific cytotoxic cells, but rather immature anergic
creasing gene expression of Tr1-related markers and associated cells [37]. Cosmi et al. considered that increased number of

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FOXP3 Treg in classical Hodgkin lymphoma
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CLINICAL RESEARCH

CD8+ in EBV-positive HL in immunocompetent patients do not find this correlation. The cHL Treg pool is probably highly het-
necessarily reflect antiviral immune response, but perhaps are erogeneous, comprising FOXP3+ and FOXP3– suppressor cells
a part of the total regulatory pool [39]. CD8 regulatory T cells among CD4 and CD8, as well as other cell types. Other possi-
share functional characteristics with CD4+ Treg; they are an- ble mechanisms maybe exist that ensure better outcome in
ergic (do not produce perforin or granzyme) and immunosup- young individuals with EBV-positive cHL.
pressive (their effectiveness in viral-induced tumors is ques-
tionable) [39, 40]. We showed that the number of FOXP3+CD8+
cells in the cHL microenvironment is variable and without sta- Conclusions
tistically significant differences. CD8+ Treg can be FOXP3-,
which could mean that the number of CD8+Treg cells was un- Epstein-Barr viral status has an impact on the composition of
derestimated by FOXP3 expression alone, and other markers the T cell population in cHL. In cHL with positive EBV status, the
are necessary to assess the CD8 Treg “pool” [20]. We found a microenvironment is more enriched with FOXP3+ Treg, mainly
decreased number of CD4 cells in the cHL microenvironment FOXP3+CD4 cells. Aging has an impact on CD4+ numbers, but
in individuals older than 26 years, which may reflect a normal does not affect the number of Treg cells in cHL.
process of immune senescence due to decreased thymic out-
put of Teff and Treg cells [40]. We did not find a difference in Acknowledgments
Treg according to age, which agrees with the results of Dejaco
and Fessler, who considered that the number of Treg cells re- The authors would like to thank Prof. Paul G. Murray (School
mains the same, but their function is changed [41,42]. In con- of Cancer Sciences, University of Birmingham, Birmingham,
trast, Raynor reported that aging is associated with increased United Kingdom) for his help and expert advice in the prepa-
number of Treg cells, with consequent increased incidence of ration of the manuscript.
tumors and autoimmune diseases [43]. Our previous study
showed that younger individuals (<35 years old) with EBV- Conflict of interest
positive cHL have longer disease-free survival [12]. We ex-
pected to find that younger individuals with EBV-positive cHL The authors declare no conflict of interest.
would have higher numbers of FOXP3+ Treg, but we did not

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