Original Paper Quantitative immunohistochemical analysis of cytokine pro®les in Epstein±Barr virus-positive and -negative cases of Hodgkin's disease D. F. Dukers 1 , L. H. Jaspars 1 *, W. Vos 1 , J. J. Oudejans 1 , D. Hayes 1 , S. Cillessen 1 , J. M. Middeldorp 1,2 and C. J. L. M. Meijer 1 1 Department of Pathology, Academic Hospital, Vrije Universiteit Amsterdam, The Netherlands 2 Bioscience Research Unit, Organon Teknika, Boxtel, The Netherlands * Correspondence to: Lies Jaspars, Department of Pathology, Academisch Ziekenhuis der Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. E-mail: eh.jaspars@azvu.nl Received: 4 January 1999 Revised: 9 June 1999 Accepted: 16 September 1999 Abstract Hodgkin's disease (HD) is a malignant lymphoproliferative disease characterized by the presence of Hodgkin±Reed±Sternberg cells surrounded by a reactive in®ltrate. In Epstein±Barr virus (EBV)-associated cases (40±60%), at least two EBV-encoded proteins [latent membrane protein 1 (LMP1) and LMP2] are expressed, which are potential targets for cytotoxic T-lymphocytes (CTLs). Although in EBV-positive cases signi®cantly more activated (granzyme B-positive) CTLs and natural killer (NK) cells are present, the cytotoxic immune response is not suf®cient for adequate killing of tumour cells. The production of immunomodulating cytokines within the tumour may be one of the mechanisms causing circumvention of the immune system. This study investigated by immunohistochemistry the presence of the immunosuppressive cytokine inter- leukin-10 (IL-10) and other Th1/Th2-associated cytokines [IL-2, IL-4, interferon-gamma (IFN-c)] in the neoplastic cells and reactive lymphocytes of nine EBV-positive and 18 EBV-negative cases of HD. The percentage of IL-10-expressing cells, both neoplastic and reactive, in EBV-positive cases was signi®cantly higher (33.1% vs. 18.5% for the neoplastic cells and 21.6% and 12.2% for the reactive cells, p=0.003 and 0.04, respectively) than in EBV-negative cases. No difference in the percentage of IL-2-, IL-4- and IFN-c-expressing cells was observed. These results suggest that escape from local immune surveillance is not due to a shift from Th1 towards Th2, but may be caused by a direct effect of IL-10 on the cytotoxic cells. Copyright # 2000 John Wiley & Sons, Ltd. Keywords: Hodgkin's disease; cytokines; interleukin-10; immunohistochemistry; EBV Introduction Hodgkin's disease (HD) is a malignant lymphoproli- ferative disorder characterized by the presence of Reed±Sternberg (R±S) cells and their mononuclear variants, the Hodgkin (H) cells, surrounded by a large number of reactive cells [1]. Amongst these reactive cells, a variable number of cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells can be found, which are considered to be part of the immune response against the neoplastic H/R±S cells. In 40±60% of cases, HD is associated with the presence of Epstein±Barr virus (EBV) in the malignant cells [2,3]. When EBV is present, the virus is tran- scriptionally active and expresses a restricted set of viral antigens, namely EBV nuclear antigen 1 and the latent membrane proteins (LMP1, 2A, and 2B) [4,5]. Both LMP1 and LMP2 have been shown to be subdominant targets for CTLs in association with different MHC class I restriction elements in vitro [6±8]. We previously found that EBV-positive cases of HD contained signi®cantly more activated CTLs than EBV-negative cases [9,10]. Moreover, EBV-positive cases were found to express relatively high levels of MHC class I molecules, suggesting that the tumour cells in these cases should be able to elicit a strong cytotoxic reaction. In spite of this, the acquired immune response is apparently insuf®cient for ade- quate killing of the tumour cells. This is presumably due to local immune suppressive mechanisms. Frisan et al. showed that tumour-in®ltrating lymphocytes of an EBV-positive case of HD failed to show EBV- speci®c cytotoxicity, whereas peripheral blood lympho- cytes of the same patient were able to do so [11]. Production of immune regulatory cytokines such as interleukin-10 (IL-10) by malignant and/or reactive cells within the tumour may be involved in this phenomenon. IL-10 can modulate the immune response at several levels; for instance, it plays a crucial role in balancing humoral (Th2-like) and cellular (Th1-like) responses by inducing a shift towards Th2 [12,13]. Direct suppressive effects of IL-10 on cytotoxic T-lymphocytes have also been described in both the human and the murine system [14±18]. Production of IL-10 in EBV-positive tumours can be expected for several reasons. For instance, LMP1 is able to induce IL-10 expression in vitro [19]. Also, EBV contains a gene, BCRF1, encoding for viral IL-10 (vIL-10), a protein highly homologous to cellular IL-10 [20,21]. Moreover, Herbst et al. [22] and Journal of Pathology J Pathol 2000; 190: 143±149. Copyright # 2000 John Wiley & Sons, Ltd. ccc 0022-3417/2000/020143±07$17.50