Immunology Letters 104 (2006) 83–88

Review

Cytokine mediated induction of the major Epstein–Barr virus(EBV)-encoded transforming protein, LMP-1

Lorand L. Kis ∗, Miki Takahara, Noemi Nagy, George Klein, Eva KleinMicrobiology and Tumor Biology Center, Karolinska Institute, S-17177 Stockholm, Sweden

Received 30 October 2005; received in revised form 30 October 2005; accepted 8 November 2005Available online 1 December 2005

Abstract

In the in vitro infected B-cells six EBV-encoded nuclear antigens (EBNA-1–6) and three latent membrane proteins (LMP-1, -2A, -2B) areexpressed (type III latency). In addition, other restricted forms of latency occur in the EBV-carrying malignancies. In Burkitt lymphoma (BL) onlyEBNA-1 is expressed (type I), while in Hodgkin lymphoma (HL), T-, and NK-lymphoma, and nasopharyngeal carcinoma EBNA-1 and LMPs areexpressed (type II). B-cells with these three expression patterns have been detected in healthy virus carriers.

While in type III latency two viral transcriptional activators, EBNA-2 and -5, are responsible for LMP-1 expression, the mechanism that controlstvCi

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he expression of LMP-1 in type II latent cells is not known. In order to study the interaction of EBV- and HL-derived cells, we studied the initro EBV-converted subline of the KMH2 cells that express only EBNA-1 and LMP-2A. Interestingly, exposure of the KMH2-EBV cells toD40-ligand and IL-4 induced LMP-1 expression, in the absence of EBNA-2. In BL cell lines lacking EBNA-2 another cytokine, IL-10, could

nduce LMP-1 expression. IL-10 induced LMP-1 also in tonsillar B-cells infected with the EBNA-2-deleted virus strain P3HR-1.Our results show that cytokines are responsible for the expression of LMP-1 in type II latent B-cells. These signals are available in the germinal

enter environment and in the granulation tissue of HLs. Based on these results we propose that LMP-1 expression is induced by extracellularignals and is not a constitutive characteristic of the EBV-carrying type II B-cells. Cytokine mediated induction of LMP-1 may also explain theeterogeneous expression of this viral gene seen in normal and malignant cells.

2005 Elsevier B.V. All rights reserved.

eywords: Epstein–Barr virus; Hodgkin lymphoma; LMP-1

. Introduction

The gamma-herpesvirus EBV is carried by 90–95% of theuman population [1]. The main target of the virus is the B-ymphocyte. The virus can infect epithelial-, T-, and NK-cells,s indicated by the occurrence of EBV-carrying neoplasms ofhese cell types [1]. In the followings, we restrict our discussiono the EBV-B-lymphocyte interaction.

EBV expresses different combinations of latency associatediral genes depending on the activation- and differentiation statef the B-cell. The two extremes are represented by the in vitroBV-infected B-cells [2] and the EBV-carrying memory B-cellsf healthy virus carriers [3]. The former are activated and trans-ormed for proliferation (lymphoblastoid cell lines, LCL), whilehe latter remain resting.

∗ Corresponding author. Tel.: +46 8 5248 67 61; fax: +46 8 33 04 98.E-mail address: Kis.Lorand.Levente@mtc.ki.se (L.L. Kis).

2. The different latency forms of EBV

In LCLs, nine EBV-encoded proteins are expressed, sixof which localize to the nucleus (EBV-nuclear antigens 1–6,EBNA-1–6), while the other three localize in the cytoplasmand plasma membrane (latent membrane protein, LMP-1,-2A, and –2B). This viral gene expression pattern is termedtype III latency [1]. In these cells, the EBNAs are expressedfrom a long polycistronic mRNA originating from promoterslocated in the BamHI C or W region of the virus genome.This broad viral gene expression is rarely seen in vivobecause such cells are eliminated by the immune response.Type III B-cells can be detected in the lymphoid tissueduring primary infection (infectious mononucleosis, IM)[4,5] and healthy virus carriers [6]. While these cells haveproliferative potential, due to the expression of immunogenicproteins and the costimulatory surface molecules they canlead to malignancy only in conditions of immuno-suppression[1].

165-2478/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.imlet.2005.11.003

84 L.L. Kis et al. / Immunology Letters 104 (2006) 83–88

Table 1EBV gene expression in latent infection of normal and malignant B-cells

EBV genes expresseda In vivo In vitro

Normal B-cell Tumor

LMP-2 (±EBNA-1) Memory B cell [3]EBNA-1 GC-B cell [9] BL, PEL [10] BL, PEL lines [11]EBNA-1, LMP-1, LMP-2 GC-B cell [12] HL [13]EBNA-1–6, LMP-1, LMP-2 Naıve B cell [6] PTLD [14] LCL, some BL lines [15]EBNA-1–6 GC-B cells in IM [5,16] PTLD [17] B-CLL [18]EBNA-1, -3, -4, -6 Rare BL [19] Rare BL lines [19]

Abbreviations: GC, germinal center; BL, Burkitt lymphoma; PEL, pleural effusion lymphoma; HL, Hodgkin lymphoma; PTLD, post-transplant lymphoroliferativedisease; LCL, lymphoblastoid cell line; IM, infectious mononucleosis; B-CLL, B-chronic lymphocytic leukaemia.

a The non-translated RNAs, EBERs, are expressed in all EBV-carrying cells.

In healthy individuals, EBV is carried by the memory B-cellsin which only transcripts of LMP-2A (termed latency 0) can bedetected [3,7]. When these cells enter cell division they expressEBNA-1 [8]. These cells are not recognized by the EBV specificimmunity.

Other restricted forms of latent gene expression showing dif-ferent assortments of the virally encoded proteins can be detectedin EBV-carrying B-cell lymphomas, in the lymphoid tissues ofIM patients and healthy virus carriers (summarized in Table 1).Burkitt lymphomas (BL) express only EBNA-1 (type I latency),while Hodgkin lymphomas (HL) express EBNA-1 and the mem-brane proteins LMP-1 and -2 (type II latency) [1]. In the germinalcenter (GC) of healthy virus carriers B-cells with types I and IIlatency have been detected [9,12]. EBNA-1 expression uses dif-ferent promoters in type I/II and type III latencies. In the formerEBNA-1 transcript initiates from the Q-promoter, while in thelatter from the W- or C-promoter [2]. EBNA-1 is instrumentalin the maintenance of the viral episome during cell division andthus its presence is pivotal in all proliferating cells.

In addition, two other forms of EBV gene expression havebeen encountered. One was first described in the in vitroEBV-infected B-chronic lymphocytic leukaemia (B-CLL) cells,where all the six EBNAs, but no LMP-1, are expressed [18].They do not have proliferative potential. Such B-cells weredetected also in the GC of tonsills and lymph nodes of IMpatients [5,16].

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cellular proteins to activate the LMP-1 and -2 promoters [2]. Thedirect partners of EBNA-2 are the repressor RBP-J kappa (alsoCBF1) (involved in the Notch signalling pathway) and PU-1(also Spi1) [2]. Both proteins bind to distinct sites in the LMP-1promoter. It is well established that LMP-1 expression in thetype III B-cells requires the expression of EBNA-2.

EBNA-2 and -5 are not expressed in latency type II cells,therefore LMP-1 is induced by a different molecular mecha-nism (EBNA-2 independent LMP-1 expression). This impliesthat other cellular or viral factors than EBNA-2 participate inthe establishment of type II pattern. EBV-carrying B-cells witha type II latency pattern have been detected in lymphoid tissuesof healthy virus carriers [1] and of IM patients. When EBV-positive, Hodgkin-, T-, and NK-lymphomas are regularly typeII [1].

Both types II and I patterns have been described in ton-sillar GC-B cells [9,12]. According to Thorley-Lawson andco-workers EBV-positive GC-B cells in healthy virus carriersexpress EBNA-1, LMP-1 and -2 mRNA [12], while Araujo etal. found that EBV-positive EBNA-2 negative GC-B cells rarelyexpress LMP-1 [9] (thus they should be type I). The impor-tance of LMP-1 expression of GC-B cells is shown in transgenicmice in which constitutive LMP-1 expression in B-cells leads tothe absence of GCs through inhibition of the BCL-6 transcrip-tion factor [23]. Repression of BCL-6 is also seen in BL cellsin which, similar to CD40-activation, LMP-1 inhibits BCL-6ecsh

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Another viral gene expression pattern was encountered inwo BL-lines, Daudi and P3HR-1. Both lines carry the Ig-c-mycranslocation and their resident virus lacks the EBNA-2 and partf EBNA-5 genes [20,21]. They express EBNA-1, -3, -4, -6, andtruncated EBNA-5 protein. Due to the absence of EBNA-2

hese viral strains do not transform B-cells in vitro. EBV-strainsith deletion in the EBNA-2 have been detected also in BLs

19].

. Regulation of LMP-1 expression

EBNA-5 and -2 are the first viral genes expressed in B-cellsnfected in vitro [2]. These proteins are transcriptional activa-ors and induce, among other genes, LMP-1 that is detected8–72 h after infection [2]. Both EBNA-2 and LMP-1 proteinsre required for the in vitro EBV-induced B-cell immortalization22]. EBNA-2 does not bind directly to DNA, it associates with

xpression [24]. However, the EBV-gene expression in EBV-arrying GC-B cells regarding LMP-1 is not clearly defined,ince existence of BCL-6 positive LMP-1-positive GC-B cellsave not been documented.

. In vitro interaction of EBV with Hodgkinymphoma-derived cells

As mentioned above, the molecular mechanism of LMP-1xpression in type II cells is not yet defined. In spite of con-iderable effort, B-cell lines with type II EBV latency have noteen established from HLs. While about 50% of Hodgkin lym-homa cases are EBV-positive, the few HL-derived cell lines areBV-negative [25]. By in vitro infection, we have establishedn EBV-positive subline of the HL-derived KMH2 cells in ordero study the EBV expression in cells that are representative forhe malignant Hodgkin–Reed Sternberg (HRS) cells. While, as

L.L. Kis et al. / Immunology Letters 104 (2006) 83–88 85

mentioned above, in vivo all EBV-positive HRS cells expressthe type II pattern, these cells expressed only EBNA-1 proteinand LMP-2A transcripts [26].

LMP-1 could be induced in the KMH2-EBV cells withchemical agents (histone-deacetylase inhibitors, phorbol ester,demethylating agent) [26], which can modulate the EBV-geneexpression in BL lines [27]. Importantly, the induced LMP-1expression occurred in the absence of EBNA-2 [26]. This indi-cates that the phenotype of the HRS cells does not allow theexpression of EBNA-2.

There is a clear difference thus between the type I latencyof BL lines and the type I converted HL line (KMH2-EBV).Though most EBV-carrying BL lines express type I latency atearly passage, usually they drift in culture to type III latency [15].In stable type I BL lines, EBNAs and LMP-1 expression can beinduced by the demethylating agent 5-azacytidine [27]. It seemstherefore that the type I latency of BL cells is determined byepigenetic regulation of the major EBV-latent promoters, whilein HRS cells the phenotype is decisive in that they do not expressB-cell specific transcription factors, such as BSAP (PAX-5) andOct-2 [28], that are known to activate the Wp/Cp promoters[29,30]. The HRS cells belong to the B-cell lineage but they arepeculiar since they lack several B-cell specific characteristics,among others surface immunoglobulin expression [31].

Similar to the KMH2-EBV cells, in the EBV-infected celllines derived from body cavity lymphoma (pleural effusion lym-pP

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In search for additional cytokines that can induce LMP-1 inthe absence of EBNA-2, we found that IL-10 induced LMP-1 inthe EBNA-2-deleted virus carrier BL lines Daudi and P3HR-1[38]. Furthermore, IL-10 induced LMP-1 expression and pro-liferation in the conditional LCL, ER/EB2-5, when EBNA-2expression was turned off [38]. Also, LMP-1 could be inducedby IL-10 even in normal B-cells when infected in vitro with theEBNA-2-deleted P3HR-1 EBV-strain [38].

6. Conclusions

We discuss now the fate of EBV infected B-cell from twoaspects. One is the connection between the B-cell differentiationpathway and EBV-gene expression; the other is the role of EBVin the development of HL.

In healthy carriers, EBV is known to reside in the memory B-cell compartment [3]; however, the stage of B-cell differentiationat which the infection occurs is unknown. Based on EBV-geneexpression profiles of sorted tonsillar B-cells Thorley-Lawsonproposed that EBV-carrying cells enter the memory B-cellpool through the antigen-induced B-cell differentiation and theexpression of EBV genes are down-regulated in certain stages[39]. According to this model, the infection of naıve B-cellsinduces the type III pattern, but when the cells enter the GC theyswitch to type II. Upon further differentiation and exit from theGC, the memory B-cells do not express EBV-encoded proteins(

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homa, PEL)-derived cell lines EBNA-2 cannot be induced [32].EL cells have also lost several B-cell characteristics [33].

Since type III latency occurs only in cells with a B-cell phe-otype [34], lack of B-cell specific transcription factors may beesponsible for the restricted EBV-gene expression in the HRSnd PEL cells.

. Induction of LMP-1 expression by extracellularignals

The characteristic feature of Hodgkin lymphoma is thathe malignant Hodgkin–Reed Sternberg (HRS) cells are scarceabout 1%) in the tissue that is made up by T-, B-cells,acrophages, eosinophils, and plasma cells. There is good evi-

ence for reciprocal interaction between the cellular componentsn which a variety of cytokines function both in an autocrinend paracrine manners [35,36]. This cytokine-ligand rich milieueems to be responsible for the survival and proliferation of HRSells. Among others, the interaction through CD40 and its liganday be of considerable importance since the HRS cells expressD40 and are surrounded by activated CD4 T-cells that expressD40-ligand [37].

We reasoned that the lack of LMP-1 expression in the in vitronfected HL cells may be due to the need for signals providedn vivo. Therefore, we exposed the KMH2-EBV cells to CD40-igand and to IL-4. Indeed we found that LMP-1 was inducedn the treated cells [26]. LMP-1 expression was maintained asong as the CD40L-IL-4 signals were provided. We succeededhus for the first time to obtain B-cells with a type II latencyn vitro; however, this viral gene expression was dependent onontinuous provision of the extracellular signals.

latency 0) [39].Both types I and II B-cells have been seen in the GC of healthy

irus carriers [9,12]. We assume that the EBV-infected B-cellsn the GC are type I. This is in line with the type I latencyf BLs, a tumor that maintains the GC-B cell phenotype. Ouresults suggest that LMP-1 expression in the type II B-cells in theC is not constitutive, but it is induced by extracellular signalsrovided by the microenvironment (Fig. 1). As long as the cellsemain in the GC and receive these signals they will expressMP-1. This is in line with the observations that EBV-positiveemory B-cells isolated from tonsils express LMP-1, while the

nes from the peripheral blood are LMP-1 negative [3,12]. Uponxit from the GC, the long-lived, resting memory B-cells down-egulate the expression of all viral proteins.

Similarly, to sustained CD40 activation in vitro [40], expres-ion of LMP-1 at the late phase of GC-B differentiation mayavour the differentiation to the memory B-cell over the plasmaell pathway. This would point to a viral strategy that utilizesot only the GC-B cell differentiation pathway but also the sig-als given by the microenvironment to enter preferentially in theemory B-cell compartment.The molecular details of LMP-2 expression in type II latency

re not known. Because of their different promoters, LMP-1nd -2 expression in type II cells may be driven by differentechanisms, such as Notch-pathway for LMP-2 and cytokines-TAT pathway for LMP-1.

Extracellular signals induce EBNA-2-independent expres-ion of LMP-1 not only in normal B-cells, but also in HL.he first step of malignant transformation is assumed to occurhen the surface Ig lacking “crippled” GC-B cells are saved byBV [35,41]. Such cells are normally destined to die by apop-

86 L.L. Kis et al. / Immunology Letters 104 (2006) 83–88

Fig. 1. Germinal center (GC) B cells and the generation of Hodgkin-Reed Sternberg (HRS) cells. The role of extracellular signals in the expression of the EBV-encodedgene LMP-1. The EBV episome is represented in the nucleus of the infected cells by a circle. sIg denotes surface immunoglobulin.

tosis (Fig. 1). The survival signals may be provided by EBVthrough its viral proteins LMP-1 and -2. These molecules areconstitutively signalling through the CD40- and BCR-pathways,respectively [2]. Recent in vitro experiments substantiate thisassumption in that EBV was shown to transform GC-B cellsthat fail to express a functional BCR [42–44].

What is the role of LMP-1 in the established malignancy? Pre-vention of apoptosis could be the initial step. LMP-1 is knownto activate the nuclear factor (NF)-kappa B survival pathway.This feature is pivotal for HL, since the NF-kappa B pathwayis activated in the EBV-negative HLs also, and the constitutiveactivation of this pathway is needed for the survival of the estab-lished HL cell lines [45]. Therefore, it seems that NF-kappaB activity is a common denominator of both EBV-positive and-negative cases. While in the EBV-carrying cases LMP-1, inthe EBV-negative cases mutations in the NF-kappa B inhibitors(IkB-alpha) [46,47], amplification of the REL gene (encodesan NF-B family member) [48], or signalling through CD30 andCD40 [49] would activate NF-kappa B. The inhibition of apopto-sis by LMP-1 could be achieved by multiple mechanisms, suchas the expression of anti-apoptotic genes, cytokines, costimu-latory surface molecules that enhance the interaction with thesurrounding cells.

LMP-2 may also contribute to the survival, since in transgenicmice it can drive B cell development and survival in the absenceof normal B cell receptor signals [50]. Furthermore, it activates

multiple signalling pathways, among others the PI3K-Akt path-way [51]. The relative importance of the different signallingpathways engaged by LMP-1 and -2 required for the survival of“crippled” GC-B cells is not known.

Elimination of EBV-carrying type III latent B-cells is criticalfor the infected individual since these cells have a proliferativepotential. LMP-1 and -2 expression together with the costimula-tory molecules and intact antigen-presenting machinery wouldbe expected to elicit an immune response against the EBV-carrying HRS cells. In the HLs, however, the immune responseseems to be inhibited locally [52] by the immuno-supressivecytokines TGF-beta and IL-10 produced by the HRS cells [53],and by the presence of regulatory T-cells [54].

Acknowledgements

Supported by funds from the Swedish Cancer Society, Swe-den. L.L.K., M.T., and N.N. are recipients of Cancer ResearchFellowship of Cancer Research Institute (New York)/ConcernFoundation (Los Angeles).

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