Immunological aspects of Epstein/Barr virus infection

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1 Critical Reviews in Oncology/Hematology 44 (2002) 203/215 Immunological aspects of Epstein/Barr virus infection Shouichi Ohga, Akihiko Nomura, Hidetoshi Takada, Toshiro Hara Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Maidashi Higashi-ku, Fukuoka , Japan Accepted 25 January 2002 Contents 1. Introduction EBV infection in the normal immunocompetent host Primary infection Acute infectious mononucleosis (IM) T cell expansion in acute IM Cytokine profile during acute IM Rare complications of acute IM Immune evasion and persistent infection Antigen presentation Apoptosis Cytokine network Genetic variation EBV infection in patients with EBV-associated diseases EBV B-cell LPD/lymphoma EBV T/NK-cell LPD (CAEBV or EBV-HLH) Conclusion remarks Reviewers Acknowledgements References Biographies Abstract Epstein /Barr virus (EBV) is a member of ubiquitous g herpes viruses, which primarily induces acute infectious mononucleosis (IM) or subclinical infection in susceptible subjects. The host reactions account for the clinical manifestation of IM. This virus also contributes to the development of lymphoid or epithelial malignancies. The outgrowth of EBV-infected B-cells is first controlled by interferon (IFN)-g and natural killer (NK) cells, and later by EBV-specific cytotoxic T-lymphocytes (CTL). To overcome the host responses and establish the persistent infection, EBV conducts the protean strategies of immune evasion. Several EBV genes modulate apoptotic signals and cytokine balances to persist B-cell infection without insulting the host. Uncontrolled lymphoproliferation occurs as EBV B-cell lymphoproliferative disease (LPD)/lymphoma in AIDS, posttransplant, or primary Abbreviations: BL, Burkitt lymphoma; CAEBV, chronic active Epstein/Barr virus infection; CTL, cytotoxic T-lymphocyte; EBV, Epstein/Barr virus; HLH, hemophagocytic lymphohistiocytosis; Ig, immunoglobulin; IFN, interferon; IM, infectious mononucleosis; IL, interleukin; LCL, immortalized lymphoblastoid cell line; LPD, lymphoproliferative disease; MNC, mononuclear cell; NPC, nasopharyngeal carcinoma; PCR, polymerase chain reaction; PID, primary immunodeficiency diseases; TCR, T-cell antigen receptor; TGF, transforming growth factor; Th, T helper; XLA, X-linked agammaglobulinemia; XLP, X-linked lymphoproliferative disease/syndrome. Corresponding author. Tel.: / ; fax: / address: ohgas@pediatr.med.kyushu-u.ac.jp (S. Ohga) /02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S ( 0 2 )

2 204 S. Ohga et al. / Critical Reviews in Oncology/Hematology 44 (2002) 203/215 immunodeficiency diseases (PID). On the other hand, EBV T/NK cells are involved in EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) or chronic active EBV infection (CAEBV) in children having no underlying immunodeficiencies, and at times lead to the clonal evolution of T/NK-cell LPD/lymphomas. Recent advance in molecular techniques has enabled us to analyze the clonality of EBV-infected lymphocytes and to quantify the gene expression of EBV and cytokines. Dominant autocrine loop of T helper (Th) 2 and Th1 may exert in EBV B-LPD and T-LPD, respectively. Intensive studies on the immunological interface between effector components and EBV target cells will provide more information on clarifying the pathogenesis of EBVassociated lymphoid malignancies, as well as on exploiting the therapeutic and preventive strategies for the formidable EBVassociated disease in childhood. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: LPD; CAEBV; Th1/Th2; Cytokine; Immune evasion 1. Introduction Epstein/Barr virus (EBV) is one of the most successful viruses, which ubiquitously infect humans and persist for the lifetime of the person [1]. Primary EBV infection is usually asymptomatic in childhood, whilst at times induces acute infectious mononucleosis (IM) in susceptible adolescents or adults [2]. During the subsequent lifelong infection, the virus carriers do not manifest symptoms as long as they are immunocompetent. EBV has evolved a successful strategy of immune evasion without disturbing the immune homeostasis of the host. On the other hand, primary and secondary immunodeficiencies provoke the virus reactivation and the excessive proliferation of EBV-infected B-cells, which lead to the development of EBV B lymphoproliferative diseases (LPD)/lymphomas [3 /7]. EBV infection in T/NKcells is involved in an array of aggressive EBV-associated diseases including hemophagocytic lymphohistiocytosis (HLH)/hemophagocytic syndrome [8,9], chronic mononucleosis/chronic active Epstein /Barr infection (CAEBV) [10 /12], and T/NK-cell lymphomas [13]. These are regarded as fulminant EBV T/NK-LPD, which affects from infants to young adults who have no apparent immunodeficiencies [14,15]. With additional environmental and genetic insults, EBV causes malignancies such as Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), and gastric cancer [2,16]. EBV may be also associated with Sjögren syndrome, systemic lupus erythematosus (SLE), rheumatoid arthritis, and inflammatory bowel diseases [17,18]. In vitro EBV has the ability to establish a latent infection in proliferating B lymphoblasts. It is the only experimental system available for studying human herpes virus latency and cellular growth mechanisms. Recently, polymerase chain reaction (PCR) technology and sophisticated cell fractionation have made a progress to unveil the in vivo behavior of the virus. EBV infection in primary immunodeficiency diseases (PID) is the experiment of nature for understanding the pathophysiology of EBVassociated diseases [19]. Analyses on the intermediary phase between the persistent silent infection and the reactivation of EBV may unravel the combating long history between the virus and humans. However, the distinctive scenario has not been completed between the exaggerating immune response in acute IM and the uncontrolled immune activation in EBV-HLH or CAEBV. We here review the immunological responses to EBV infection in normal immunocompetent subjects, and in patients with EBV-associated diseases. The immune perturbation by EBV was focused on the cytokine balance affecting both host immune response and viral evasion system. 2. EBV infection in the normal immunocompetent host 2.1. Primary infection EBV is an enveloped g herpes virus which contains double strand linear DNA of 170 /175 kb in the nucleocapsid [1]. This virus enters oropharynx and adjacent structures, and preferentially infects B-cells via the C3d complement receptor, CD21. Primary infection during early childhood is mostly asymptomatic, and that during adolescence causes acute IM in 30/50% of cases [2]. Despite the rule of subclinical early infection in developing countries, more than 80% of Japanese children acquire primary infection until 3 years of age. It remains unknown whether the age-related presentation depends on the initial viral load or host immunity. Salivary transmission of EBV-infected B-cells seems to be the main route of infection. The infectivity of cell free viral DNA is obscure. The initial productive infection in epithelial cells is unlikely. EBV replicated in cultured squamous epithelial cells, and EBV /DNA was detected in desquamated tonsillar epithelium [20]. IgAmediated infection and excretion of EBV in oropharyngeal epithelial cells were indicated [21]. Nevertheless, X-linked agammaglobulinemia (XLA) patients were seronegative for EBV infection and showed no EBVspecific T cell immunity [22]. They originally lack of mature B-cells expressing CD21 but sustain normal T cell immunity. This observation suggests that CD21 B- cells are essential for the primary infection of EBV. The epithelial lining is discontinuous that allows direct access of incoming virus to underlying tonsillar B-cells. Several molecules other than CD21 are the candidates of

3 S. Ohga et al. / Critical Reviews in Oncology/Hematology 44 (2002) 203/ EBV receptor [23]. After the initial attachment of viral gp350/220 and cellular CD21, the post-binding interaction between the viral gp25/gp85/gp42 complex and MHC class II molecules (HLA-DR, DP, DQ) might mediate internalization of the virus [24]. On the other hand, bare lymphocyte syndrome patients have been infected with and transformed by EBV despite the complete lack of MHC class II expression [24]. CD21 is the major receptor of EBV, although coreceptor system or CD21-independent infection route may exist. In vitro EBV infection of B-cells gives rise to the immortalized lymphoblastoid cell lines (LCLs) that have been analyzed as a representative model of latencyinfected cells in normal humans. LCL is continuously activated and proliferate to express 6 nuclear antigens, EBNA1, 2, 3 (3A), 4 (3B), 5 (LP), and 6 (3C), 3 latent membrane proteins (LMP)1, LMP2A, and LMP2B, along with three kinds of untranslated RNAs, EBVencoded mrna (EBNA)1, EBNA2, and a family of BamH1 A transcripts [15,25]. EBERs are small RNAs without having polya tail. The BamH1 A transcripts from BARF0 are translated in lytic, but not in latent phase. LCL shows high expression of Fas, but is resistant to Fas-induced apoptosis. BZLF1 and BRLF1 are the immediate early genes of lytic infection. BMRF1, BALF2, BHRF1 and BCRF1 are the early lytic genes. Glycoprotein-encoding genes including BARF4 (encodes gp110) and BLLF1 (encodes gp350) are the late genes. Following the initial replicative (lytic) infection, EBV genome circulizes to be maintained as a multicopy plasmid in the B-cell nucleus. The vast majority of EBV-infected B-cells is latent and has the capacity to induce immortalization. In vivo latent EBV B-cells include (1) immunoblastic cells which are highly immunogenic and rapidly eliminated during acute IM, (2) memory cells which progressed through the normal pathway of B-cell differentiation and localized in lymphoid follicles, and (3) resting nonimmunogenic cells as the latent virus reservoir in the circulation of healthy carriers. LCL belongs to latency 3 that expresses all nine proteins and three transcripts of EBV latent genes (Table 1). On the other hand, in vivo EBV memory B-cells show the restricted gene expression (EBNA1, LMP1, LMP2A, EBNA2 3 ) as seen in a latency 2 of Hodgkin disease, NPC, or CAEBV [26]. EBV B-cells lead to both latent and lytic replication in tonsils, and the produced virus expands to the adjacent B-cells [27] Acute infectious mononucleosis (IM) Acute IM patients present with fever, painful lymphadenopathy, sore throat with pharyngotonsillitis, hepatosplenomegaly, and skin eruption. More than 80% of cases with acute IM are due to primary EBV infection. Puffy eyelids and tonsillar exudates are characteristic for IM due to EBV infection. Younger children appear to have higher rates of skin eruptions and abdominal pain than young adults. After the incubation period of 2/7 weeks, EBV-infected B-cells increase to 1 /20% of circulating B-cells of adolescents or young adults during acute IM [2]. The proliferation of EBV-infected B-cells is quickly abrogated in the first 2 weeks of IM by a brisk cellular responses comprising natural killer (NK) cells, interferon (IFN)g, activated CD8 T cells, and antibody-dependent cell-mediated cytotoxicity (ADCC) [28]. The activated CD8 T cells, morphologically defined as atypical lymphocytes, at Table 1 Cellular and viral phenotype in types of EBV latency Latency Type 1 Type 2 Type 3 Viral expression EBNA1 EBNA1 EBNA1 BARF0 BARF0 BARF0 EBERs EBERs EBERs LMP1 and LMP2 LMP1 and LMP2 EBNA2/6 Cellular expression CD10, CD77 CD23, CD80, CD86, CD40 CD23, CD80, CD86 TAP-1/2 low TAP-1/2 TAP-1/2 LFA1/3, ICAM1 LFA1/3, ICAM1/2 HLA class I HLA class I In vitro CTL response Resistant Susceptible Susceptible EBV-infected cells Burkitt s disease Hodgkin s disease PTLD Gastric ca. Nasopharyngeal ca. AIDS-related lymphoma Nasal T/NK cell lymphoma Oppotunistic lymphoma CAEBV PAL, LCL EBNA, Epstein/Barr virus nuclear antigen; BARF, BamH1-A rightward reading frame; EBERs, Epstein/Barr virus encoded mrnas; LMP, late membrane protein; TAP, transporter associated with antigen processing; LFA, lymphocyte function-associated antigen; ICAM, intercellular adhesion molecule; HLA, human leukocyte antigen; CAEBV, chronic active Epstein/Barr virus infection; PTLD, post-transplant lymphoproliferative disorder; AIDS, acquired immunodeficiency syndrome; PAL, pyothorax-associated lymphoma; LCL, lymphoblastoid cell line; ca., carcinoma.

4 206 S. Ohga et al. / Critical Reviews in Oncology/Hematology 44 (2002) 203/215 times occupy more than 60% of peripheral mononuclear cells (MNCs) at the full symptoms of acute IM [29]. In the symptomatic phase, copies of EBV/DNA are detected in peripheral MNCs or serum by quantitative real-time PCR [30,31]. The flow cytometric (FACS) analysis (the increased number of HLA-DR CD8 T cells and the reduced ratio of CD4/CD8) and the quantification of EBV genome are helpful for the early diagnosis of EBV-induced IM. Changes in titers of EBV-specific antibodies confirm the diagnosis, while IgM responses to EBV/VCA are poor in infants. The immune responses reduce the number of circulating EBV B-cells by about 10 4 times, to less than one infected cell per 10 6 B-cells, within 4/6 weeks T cell expansion in acute IM The selective expansion of Vb6 and Vb7 subsets were reported in some IM patients [32]. This phenomenon was supposed to be driven by EBV-induced superantigens [33], which explained the exaggerated response by proinflammatory cytokines during acute IM. However, recent studies demonstrated that the clonally expanded CD8 T cells with skewing TCR Vb usages were driven by the conventional EBV-derived peptides rather than the EBV-induced superantigens [34]. The increased T-lymphoblasts during acute IM show activated phenotype (HLA-DR, CD38, CD45RO, CD62L ). T cells specific for lytic epitopes accounted up to 44% of the CD8 T cell pool, while CTL specific for the immunodominant proteins of EBNA3, EBNA4, and EBNA6 reached only 1/2% [35,36]. FACS analysis on the autologous LCL stimulated cells defined the bulky expansion (30 /60%) of IFNg expressing class I- restricted activated CD8 T cells, irrespective of the host HLA type [37,38]. Thus, the brisk responses to EBV in acute IM mainly result from the expansion of the lytic epitope-specific CD8 T cells. Recent intensive works on acute IM have identified a range of CTL specific epitopes within lytic EBV proteins including the immediate early (BZLF1, BRLF1), the early (BMLF1, BHRH1, BMRF1, BARF2) [39 /42], and the late proteins (gp350, gp85, gp110) [43]. The strong ex vivo CTL reactivities are induced to the late lytic antigens of some glycoproteins. EBV-specific CD4 CTL activity can be recovered from acute IM patients. The CTL clone recognizes an epitope from BHRF1 [44]. BHRF1-specific CD3 CD4 CD8 clones were also recovered [39]. EBVspecific CD4 T cells may play a significant role in primary infection and in the maintenance of functionally competent CD8 memory [45]. gdt cell can expand during acute IM [46]. Vd1 T cells proliferated to response to EBV-transformed B-cells [47,48]. Although the gdt cells might be cytotoxic against EBV-infected cells [49], the significance and the substantial magnitude of in vivo immune responses remain elusive Cytokine profile during acute IM Cytokines play a cardinal role in the pathophysiology of acute IM. Elevated levels of interleukin (IL)-1a, IL-2, IL-6 and IFNg have been detected in patients sera with acute IM [50,51]. The cytokine profile mainly depends on the expansion of activated CD8 T cells, although other immune effectors (CD4 T cells, NK cells, monocytes, neutrophils, and epithelial cells) as well as the targets (EBV-infected B-cells) release cytokines (Fig. 1). In IM tonsils, lymphotoxin (LT), tumor necrosis factor (TNF)a, and IL-6 were predominantly expressed in EBV-infected cells [52]. On the other hand, IL-1a, IL- 1b, and IL-8 were not expressed in EBV-infected cells, but did in neighboring EBV-negative cells from interfollicular area. Spender et al. [53] reported that LT, TNFa, granulocyte colony-stimulating factor (G-CSF) were expressed in EBV-infected B-cells and regulated by EBNA2. Lotz et al. [54] revealed that IFNa was released from NK cells and B-cells within 24 h of infection, while IFNg was exclusively secreted from T cells in the presence of IL-1 and IL-2, at the maximum 8 days after infection. The source of IL-1 was NK cells and monocytes. Setsuda et al. [55] demonstrated the gene expression of IL-18, IFNg, monokine induced by IFNg (Mig), and IFNg inducible protein-10 (IP-10) in tonsils of EBV-induced IM. Steigerwald-Mullen et al. [56] showed that EBNA1-specific CD4 T cells preferentially produced Th2 type cytokines (IL-5) in response to antigenic stimulation. By contrast, Bickham et al. [57] revealed the polarized Th1 phenotype (IFNg) of EBNA1-specific CD4 T cells, suggesting their resistant functions to EBV infection and EBV-associated malignancies. It remains unknown to what extent these results account for the in vivo cytokine dynamics during acute IM. EBV can also interact with neutrophils and monocytes. This virus binds to the surface of monocytes, and activates the gene expression of IL-6 but inhibits that of TNFa [58,59]. EBV modulates the IL-1 balance of neutrophils in favor of the production of IL-1 receptor antagonist (IL-1Ra) [60], and simultaneously induces neutrophil apoptosis [61]. The EBV binding effects further produce IL-8 and macrophage inflammatory protein-1a (MIP-1a), the syntheses of which are enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF) [62]. Conversely, GM-CSF has been known to facilitate the spontaneous outgrowth of EBVinfected B-cells from acute IM patients [63]. Taken together, EBV-induced chemokine production is enhanced by GM-CSF, and may serve to potentiate a host defense mechanism directed toward the eradication of virus, but simultaneously to enhance the ability to infect lymphocytes via increased recruitment to site of infections. EBV might have evolved to modulate the cytokine network in order not to be completely eliminated after primary infection.

5 S. Ohga et al. / Critical Reviews in Oncology/Hematology 44 (2002) 203/ Fig. 1. Cytokine profiles in (A) primary infection of EBV or (B) EBV-associated LPD. LT, lymphotoxin; IFN, interferon; TNF, tumor necrosis factor; vil-10, viral interleukin-10; Mo/Mf, monocyte/macrophage; TGF, transforming growth factor Rare complications of acute IM The vast majority of IM patients remit without sequelae, while a wide range of complications has been reported [64]. Splenic rupture and airway obstruction occur in B/0.5 and B/5% of adult cases, respectively. Both are precipitated by the lymphocyte expansion in the affected organs; reactive T cells in spleen, and EBV B immunoblasts in tonsils. More than half of patients show ampicillin and b-lactam antibiotics-related skin eruption. While neutropenia and thrombocytopenia are relatively common, pancytopenia is a grave complication, especially in preceding X-linked lymphoproliferative syndrome (XLP) or fatal IM. Central nervous system (CNS) complications occur in 1 /5% of patients, including cerebellitis, meningoencephalitis, cranial neuritis, transverse myelitis, Guillain/Barre syndrome. Fulminant hepatitis is very rare. The mechanism of complications has not been solved Immune evasion and persistent infection Circulating IgD B-cells are the memory B-cell pool with the long-lived persistent EBV infection [65]. In healthy carriers, the viral load is stable over time at around 1/50 EBV-infected cells per 10 6 circulating B- cells [25,66]. The B-cells harboring the episomal EBV genes of latent form are intermittently or continuously secreted as infectious virus into saliva. Classical CD8 HLA-class I restricted T cells rather than CD4 T cells show the dominant response in carrier state [67]. The frequency of EBV-specific T cells varies according to the methodology including limiting dilution analysis, enzyme-linked immunospot assay (ELISPOT), FACS staining with tetrameric MHCpeptide complexes [42]. FACS analysis on IFNg expressing T cells against autologous LCL or EBV lysate, displayed /1% of EBV-specific CTL circulate in the periphery of EBV-seropositive healthy carriers [37,68]. EBV /CTL in the blood of long-term healthy carriers are predominantly small lymphocytes with resting phenotype (HLA-DR, CD38, CD62L ) [25,35]. Many CTL target epitopes have been defined in class I restricted manner (i.e. HLA A8-restricted FLRGRAYGL of EBNA3). In persistent infection, major latent epitopes targeted by CTL are the EBNA3 family of EBNA3 (3A), EBNA4 (3B), and EBNA6 (3C). In contrast to the expression of nearly 100 viral genes during replication, EBV B-cells express at most 12 latent genes. The limited numbers of viral genes expressed reduce the opportunity to permit the recognition of infected cells by CTLs. EBNA1 proteins bind to the orip, and promote the replication of circular episome by host cell DNA polymerase. EBNA1 proteins up-regulate the expression of recombination activating genes (RAG) 1 and 2. EBNA2 proteins induce B-cell Table 2 Potential mechanisms of immune evasion by EBV gene/gene products Antigen presentation EBNA1 a Inhibited by GAR BZLF2 b Blocks the presentation of class II epitope Apoptosis BHRF1 b Homologous to Bcl2 LMP1 a Homologous to CD40 (TNF receptor family) Cytokine balance BCRF1 b Homologous to human IL-10viral IL-10 EBI3 b Complexed with the p35 subunit of IL-12 BARF1 b Decoy receptor of colony stimulating factor (CSF) 1 LMP1 a Induces IFN regulatory factor 7 Genetic variation of the virus EBNA2 a A (type 1), northern hemisphere B (type 2), southern hemisphere EBNA4(3B) a A selective mutation in the epitope recognized by HLA- A11-restricted CTL LMP1 a A 30-bp depletion mutant isolated from EBV neoplasms a Latent genes. b Lytic (replicative) genes.

6 208 S. Ohga et al. / Critical Reviews in Oncology/Hematology 44 (2002) 203/215 proliferation by up-regulating the expressions of CD21, CD23, and LMPs, or by interacting with DNA binding nuclear proteins. EBNA3 family proteins contribute to B-cell immortalization by up-regulating the cellular genes, and inhibiting the EBNA2 function to activate LMP2. LMP1 is an oncogene, which induces a signaling response mimicking a constitutively active form of CD40. LMP1 binds to several TNF receptor-associated factors (TRAFs), which result in the up-regulation of Bcl2, nuclear factor (NF)-kB, cellular adhesion molecules, cytokine productions and B-cell proliferation. LMP2 blocks the tyrosine kinase phosphorylation and prevents reactivation from latency. From another point of view, the latent or lytic genes/ gene products affect the host immune response by the mimicking function of cellular molecules to maintain the long-term infection in resting B-cell pool. Like other herpes viruses, EBV has evolved immunomodulatory mechanisms to evade the host immunity. The immune perturbations are involved in (1) antigen presentation, (2) apoptosis, and (3) cytokine network, and (4) genetic variation (Table 2) Antigen presentation Some EBV-encoded proteins are known to perturb the endogenous processing and presentation of CTL epitopes. EBNA1 is resistant to CTL-mediated immune recognition, as compared with EBNA3 family. It is mediated by an internal glycine /alanine repeat (GAR) domain that protects EBNA1 from entering the HLAclass I pathway of antigen presentation by inhibiting the proteolytic degradation through the proteasome [69]. While, the isolation of CD4 or CD8 EBNA1-specific CTL from healthy carriers implies the processing and presentation of EBNA1 epitopes via a proteasomeindependent cross-priming pathway [70,71]. Spriggs et al. [72] showed that BZLF2 protein binds to the b chain of MHC class II, and blocks the presentation of class IIrestricted CD4 T cell epitope. Anti BZLF2-specific antibodies further prevent the infection of MHC class II positive B-cells [73]. BZLF2 may have a dual function, modulating class II-restricted antigen presentation and facilitating infection of class II-positive cells. By contrast, a converse example is BNLF1 (LMP1 gene). BL cells showed the low expression of transporter associated with antigen processing (TAP) 1 and 2, and HLA class I, that was reversed by LMP1 alone [74] Apoptosis At least two EBV-proteins inhibit apoptosis [75]. BHRF1 encodes a 17 kda protein with both sequence and functional homology with the anti-apoptotic Bcl-2 oncogene. BHRF1 can protect BL cells or epithelial cells from apoptosis induced by TNFa, anti-fas, and serum deprivation [76]. LMP1 expression in B-cells induces the up-regulation of cell surface activation antigens (CD23 and CD40) and adhesion molecules (CD54 and lymphocyte-function-associated antigens (LFA) 1 and 3), and is associated with protection from cell death via induction of anti-apoptotic proteins such as A20 and Bcl-2 family. These may support that both lytic (BHRF1) and latent (LMP1) genes have the ability to enhance the survival of EBV-infected cells in vivo Cytokine network Many viruses encode novel homologs of cytokines. The viral cytokines are proposed to act as the negative and positive regulators of antiviral immune responses. BCRF1 exhibits 78% identity to the deduced amino acid sequence of human IL-10, as called as viral IL-10 (vil10). BCRF1 proteins negatively regulate IL-12 to inhibit IFNg production. This protein abrogates the T cell function to inhibit the outgrowth of EBV-infected B-cells, through suppression of IL-1 or IFNg-mediated T cell activation, or CD28 or CTLA-4 [77]. EBVinduced gene 3 (EBI3) encodes a 34 kd glycoprotein that accumulates in the endoplasmic reticulum. EBI3 is associated with the p35 subunit of IL-12, which suggests that EBI3-p35 heterodimer may be an important modulator of IL-12-dependent cell-mediated immunity [78]. BARF1 proteins exert as a soluble receptor for colony-stimulating factor (CSF) 1 [79]. Since CSF1 usually enhances the expression of IFNa in monocytes, BARF1 protein may exert as a decoy receptor to block the action of the cytokine [80]. As IFNg and IFNa inhibit the outgrowth of EBV-infected cells, these proteins may help the virus to evade the host immune system during acute IM or reactivation of virus from latency-infected cells. LMP1 could induce the production of IL-6, IL-8, and IL-10 [81 /83]. LMP1 also induces IFN regulatory factor (IRF)-7 that may be relevant to the silencing of Qp in EBV type 3 latency [84]. The overall biological effects of LMP1 on the cytokine balance in EBV infection remains elusive Genetic variation There are two major EBV strains of type 1 (A) and 2 (B) based on the EBNA2 gene polymorphism. EBV subtypes detected in EBV-associated tumors seem to reflect the background frequency in healthy carriers from the same geographical regions. Intratypic strain differences were supposed to influence on CTL recognition. EBV isolated from Southern Asia where HLA-A11 is common, had a unique polymorphism within the HLA-A11-restricted epitope of EBNA4 gene. EBNA4 expressing cells are usually recognized by HLA-A11- restricted CTLs, while the CTL response does not occur against the cells with the mutated EBNA4 antigens. It was noted as a representative evasion of EBV from host immunosurveillance. However, similar mutations within the epitope were found in the area where HLA-A11 is extremely rare. The viral polymorphism might not be

7 S. Ohga et al. / Critical Reviews in Oncology/Hematology 44 (2002) 203/ the result of CTL-induced pressure to escape immune recognition [85]. The higher tumorigenic potential of the 30-bp deleted LMP1 variant for EBV-associated neoplasms is on controversy [86]. 3. EBV infection in patients with EBV-associated diseases EBV is associated with various malignancies and autoimmune diseases. NPC and pyothorax-associated lymphoma (PAL) are reported mainly in Asian adults. EBV-associated BL is rare in Japan. The most problematic EBV-associated diseases in childhood are EBV- HLH, CAEBV and EBV LPD/lymphoma. In contrast to opportunistic lymphoma in AIDS or posttransplant patients, the EBV-associated lymphoproliferation can originate from T/NK cells. CAEBV is a chronic mononucleosis, characterized by the constellation of fever, cytopenia, hepatosplenomegaly, along with the clonal proliferation of EBV [10/12]. This syndrome may be a selective immunodeficiency to EBV infection, but most cases are sporadic, apart from XLP [87]. EBV-HLH is a secondary hemophagocytic syndrome characterized by fever, cytopenia, hepatosplenomegaly, and disseminated intravascular coagulation [8,9]. The primary form, familial HLH (FHL) is an inherited disorder [88,89]. About 20% cases with FHL are due to perforin defects [90/92]. Hypercytokinemia is a hallmark of primary and secondary HLH, and it well explains the clinical manifestation and pathophysiology of the disease [93 / 95]. Recent advances in molecular techniques have made a success to determine the localization, clonality, and quantification of EBV genome in EBV-associated diseases. Based on the cell type of EBV-infection, EBV- HLH and CAEBV has been classified into EBV T/ NK-LPD [13,14]. Kasahara et al. [96] have recently indicated the differential cellular target of EBV-infection; predominant CD8 T cells in EBV-HLH, and non-cd8 lymphocytes in CAEBV. However, EBVinfection could extend to various types of cells in an affected individual with CAEBV. The critical issue is the origin of EBV T/NK cells in EBV-HLH or CAEBV. The mechanism and significance of EBV infection to non-b-cell populations are unknown [61,97]. Immature thymocytes express appreciable levels of CD21, the EBV-receptor [98]. Clonal proliferation of EBV-infected T cells is evident in EBV/HLH and CAEBV. These may raise the possibility that EBV can infect T cells at the early developmental stage, and clonally expand in EBV- HLH or CAEBV. Patients with EBV T/NK-LPD might have a predilection to allow the selective advantage of EBV-infected T/NK cells after primary EBV infection EBV B-cell LPD/lymphoma LPD is an ominous complication arising from the outgrowth of EBV-infected B-cells as seen in PID, AIDS, therapy-related immunosuppression, and posttransplantion [3/7]. EBV B-LPD generally develops in PID with T cell deficiency, but not in XLA (Table 3). On the other hand, EBV-HLH and CAEBV are closely associated with EBV T/NK cell lymphomas. Posttransplant LPD (PTLD) stems from donor B-cells and tends to be aggressive after allogeneic hematopoietic stem cell transplantation (HSCT), while it also derives from recipient B-cells and leads to an indolent course after organ transplantation. Early diagnosis of PTLD is difficult because of the extranodal onset and no specific symptoms. Prophylactic infusion of EBV-specific ornonspecific donor lymphocytes may be efficacious. However, EBV /CTL infusion was not effective for the treatment of PTLD infected with EBNA3B deletion mutant [99]. The risk of graft-versus-host disease Table 3 Immune responses to EBV infection in immunodeficiencies Specific Ab EBV/CTL NK activity T cell deficiency a High Nonormal? Lownormal Combined immunodeficiency b Nolow No Nonormal XLP primary infection Low No Normal XLP long-term survivors Low No No CAEBV High Low Nonormal XLA Not susceptible to primary infection of EBV Posttransplant Normalhigh No Nonormal AIDS High No Nonormal Healthy virus carrier Normal Normal Normal XLP, X-linked lymphoproliferative disease; XLA, X-linked agammaglobulinemia; AIDS, acquired immunodeficiency syndrome; CTL, cytotoxic T-lymphocyte (revised from [19]). a T cell deficiency includes ataxia telangiectasia, Wiskott/Aldrich syndrome, Chediak/Higashi syndrome. b Combined immunodeficiency includes severe combined immunodeficiency and common variable immunodeficiency.

8 210 S. Ohga et al. / Critical Reviews in Oncology/Hematology 44 (2002) 203/215 (GVHD) must be overcome. Sequential monitoring of viral load in whole blood [100,101] and/or MNCs [30] is useful for predicting the development of PTLD. The transplantation from EBV-seropositive donor to EBVseronegative recipient raises the risk of PTLD. Cord blood (CB) might an ideal stem cell source to avoid EBV-associated complications. Nevertheless, EBV B- cell PTLD are reported in both EBV-seropositive [102] and EBV-seronegative recipients after CB transplants [103]. The latent EBV B-cells proliferate during the development of PTLD. Endogenous expansion of CD8 T cells including EBV/CTL leads to the spontaneous regression of PTLD [104]. The cytokine profiles are shaped by the effector cells as well as target tumor cells, and may influence on the tumor growth. The cross-linking of CD21 by gp350/220 results in increased amounts of IL-6 mrna and protein [105]. The IL-6 up-regulation may be involved in the activation of NF-kB in the lymphocytes [106]. In primary EBV infection to normal subjects, monocyte-derived IL-6 regulates B-cell growth and differentiation in a paracrine fashion. On the other hand, IL-6 exerts as an autocrine growth factor for EBV-immortalized B-cells [107]. IL-6 promotes the growth of EBV B-LPD and anti-il-6 antibodies decrease the incidence of the tumor, suggesting the autocrine loop of IL-6 for the progression of PTLD [108]. Setsuda et al. [55] reported that IFNg or TNFa was expressed in lymph nodes of patients with IM, but not, in those with PTLD. Cytokine profile in PTLD has a tendency of Th 2 predominance [109,110]. Nalesnik et al. [111] defined a cytokine pattern of IL- 2, IFNg, IL-4, and IL-10 in specimens of PTLD by using semi-quantitative RT-PCR, and suggested that LPD exists within a Th2 like cytokine microenvironment. Yao et al. [112] proposed the pivotal role of IL-18 as an endogeneous inducer of IFNg expression, along with IL-12 and IP-10, for the regression of EBV BL. The Th2 polarization in EBV LPD may reduce the host response of EBV /CTL and induce a growth advantage in autocrine fashion (Fig. 1B). On the other hand, Johannessen et al. [113] have revealed the expression of IL-2, IL-4, IL-6, IL-10 and IFNg in human B- LPD developed in severe combined immunodeficiency (SCID) mice. In the experiments, the cytokine pattern of B-LPD was notably mimicking that of activated T cells, and the complete T cell depletion hindered the tumor formation. Preconditioning regimen using T cell depletion and/or anti-thymocyte globulin (ATG) is the major risk factor for PTLD. However, T cells might be essential for the initial growth of EBV B-cells, and the established tumor could sustain its growth in an autocrine, cytokine-stimulated manner, independent of T cell help EBV T/NK-cell LPD (CAEBV or EBV-HLH) The production of IL-1b and IL-6 was depressed, but that of IFNg and IL-12 was exclusively higher in MNC from EBV-seropositive donors [114]. The cytokine response of T cell-depleted MNC was similar between EBV-seropositive and EBV-seronegative subjects, which suggested that EBV specific memory T cells are the major source of IFNg and IL-12 production against EBV reactivation (Fig. 1A). On the other hand, there are a limited number of reports on the cytokine profile of EBV-infected T cells. Groux et al. [115] established EBV T cell lines, all of which secreted IL-2. EBV promotes the proliferation of immature thymocytes in an IL-2 mediated response [116]. EBV-infected T cells can up-regulate TNFa production [117]. Roncella et al. [118] reported that EBV T cell lymphoma expressed Th1 type (IL-2 and IFNg) as well as proinflammatory (TNFa and IL-6) and immunosuppressive (transforming growth factor (TGF)-b1) cytokines. The overexpression of Th1 type cytokines (IL-18, IL-12 and IFNg) plays a pivotal role in the pathophysiology of EBV- HLH [93 /95]. Mig and IP-10 are expressed and contribute to the pathogenesis of tissue necrosis and vascular damage associated with EBV LPD [119]. The cytokines released from EBV T cells in CAEBV may express the clinical phenotype such as HLH, and induce a growth advantage of EBV-infected T cells [55,117]. Skewed TCR repertoire may be involved in the persistent activation of T cells in CAEBV [120]. Although cytokine profiles and T cell repertoire should be compared between EBV T cells and EBV reactive T cell populations, autocrine loop of Th1 cytokines may exist in the persistent reactivation of EBV (Fig. 1B). IL-10 is an autocrine factor for the proliferation of LCL, and B-cell lines established from PTLD patients produce IL-10 [121,122]. Conversely, IL-10 enhances T cell cytotoxicity against EBV-infected cells in vitro [123]. Circulating vil-10 is elevated in CAEBV patients [124]. The restricted expression of the latency associated EBV genes and the production of vil-10 and Bcl homologue may favor the growth of cutaneous EBV NK/T cell lymphomas [125]. In contrast, Shen et al. [126] reported that EBV nasal NK/T-cell lymphomas expressed human IL-10, but not, vil-10. Cytokines may contribute to the tumor growth in a distinct manner between EBV carcinoma and lymphoma [127,128]. The different expression patterns of NK inhibitory receptors are suggested between EBV NK leukemia and EBVassociated hypersensitivity to mosquito bite (HMB), the majority of which are associated with NK type CAEBV [129]. The cytokines within the tumor may lead to the circumvention of immune system, although the deranged pattern may depend on cell types and differentiation stages of EBV-infected cells. TGF-b regulates the proliferation and differentiation of cells, and angio-

9 S. Ohga et al. / Critical Reviews in Oncology/Hematology 44 (2002) 203/ Fig. 2. Progressive immune perturbation in Epstein/Barr virus-infected lymphocytes. EBV, Epstein/Barr virus; NK, natural killer; CTL, cytotoxic T-lymphocytes; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; MIG, monokine induced by IFNg; IP-10, IFNg-inducible protein-10; LPD, lymphoproliferative disease. genesis. Fahmi et al. [130] reported that TGFb-1 induces BZLF1 expression by an indirect mechanism. EBV activation from latency is initiated by the expression of ZEBRA, the protein product of BZLF1. Xu et al. [131] reported that high serum levels of TGF-b in EBVassociated diseases positively correlated with EBVspecific IgA titers. The remarkable expression of TGFb in CAEBV seems to account for the persistent reactivation of the virus [31]. Comparable high expression of IL-10 may also imply a potential inclination to Th2 state in CAEBV [132]. However, Dukers et al. [133] suggested that escape from local immune surveillance is not due to the shift from Th1 towards Th2, but may be caused by a direct effect of IL-10 on the cytotoxic cells. Otherwise, the expressions of both IL-10 and TGF-b may reflect the regulatory T cell-like responses. Low IL- 10-producing capability could make individuals more susceptible to aggressive EBV infection [133]. The vulnerability to EBV-diseases may be associated with the individual difference in the cytokine producing ability due to the polymorphic cytokine associated genes. 4. Conclusion remarks In this decade, molecular techniques have made a progress to verify the functional property of EBV /CTL for recognizing peptide in class I-restricted manner. Quantitative and quantitative analyses on EBV-infected lymphocytes and viral load have been available. The Th1/Th2 polarization of cytokine balance has been studied in EBV infection. However, there remains unknown to what extent EBV T cells may contribute to the CTL response in CAEBV. Although EBV T/ NK LPD may be one of the genetic disorders with defective EBV /CTL, SAP gene abnormalities do not explain the susceptibility to EBV in XLP patients. The immunological interface between effector components and EBV target cells should be more clearly delineated (Fig. 2). Further characterization of the individual EBVinfected T cells may shed some light not only on the mechanisms of clonal evolution of lymphoid malignancies, but also on the therapeutic and preventive strategies for the EBV-associated diseases [134]. Reviewers Shinsaku Imashuku, Kyoto City Institute of Health and Environmental Sciences, 1-2 Higashi-Takada-cho, Mibu, Nakagyo-ku, Kyoto , Japan. Ih-Jen Su, Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan, ROC. Rajiv Khanna, Tumor Immunology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, 300 Herston Road, Herston, Qld. 4006, Australia. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research (C) to Ohga S. from the Ministry of Education, Science, Sports and Culture of Japan. We wish to thank Dr Naohiro Suga (Department of Pediatrics, Graduate School of Medical Science, Kyushu University) for helpful discussions.

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