Epstein-Barr Virus (EBV) Load and Cytokine Gene Expression in Activated T Cells of Chronic Active EBV Infection

1 Epstein-Barr Virus (EBV) Load and Cytokine Gene Expression in Activated T Cells of Chronic Active EBV Infection Shouichi Ohga,1 Akihiko Nomura,1 Hidetoshi Takada,1 Kenji Ihara,1 Kiyoshi Kawakami,3 Fumio Yanai,2 Yasushi Takahata,1 Tamami Tanaka,1 Naoki Kasuga,4 and Toshiro Hara1 1 Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, and 2Department of Pediatrics, School of Medicine, Fukuoka University, Fukuoka, 3Division of Pediatrics, Kagoshima City Hospital, Kagoshima, and 4Otsuka Assay Laboratories, Otsuka Pharmaceutical, Tokyo, Japan Epstein-Barr virus (EBV) is a ubiquitous g-herpesvirus that is associated with various malignancies, including Burkitt’s lymphoma, Hodgkin’s or non-Hodgkin’s lymphoma, nasal T cell lymphoma, pyothorax-associated lymphoma, and nasopharyngeal, thymic, or gastric carcinoma [1, 2]. Primary infection with EBV is usually asymptomatic but, at times, induces acute infectious mononucleosis in susceptible immunocompetent subjects. The outgrowth of EBV-infected B cells is controlled first by interferon (IFN)–g and NK cells and later by EBV-specific cytotoxic T lymphocytes [3, 4]. The host reaction explains the clinical symptoms of acute infectious mononucleosis. On the other hand, cellular immunodeficiency elicits the persistent reactivation of EBV and the subsequent development of B cell lymphoproliferative disease [5, 6]. Chronic active EBV infection is a rare chronic mononucleosis of unknown cause, which is characterized by the constellation of fever, cytopenia, and hepatosplenomegaly, as well as the the clonal proliferation of EBV [7–10]. This syndrome is considered to be a selective immunodeficiency to EBV infection, but most cases are sporadic, apart from X-linked lymphoproli- Received 19 June 2000; revised 18 September 2000; electronically published 21 November 2000. Informed consent was obtained from parents or guardians. Financial support: Grant-in-aid for scientific research (C) (to S.O.) from the Ministry of Education, Science, Sports, and Culture of Japan. Reprints or correspondence: Dr. Shouichi Ohga, Dept. of Pediatrics, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan (). The Journal of Infectious Diseases 2001; 183:1–7 q 2001 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2001/18301-0001$02.00 ferative disease [10, 11]. EBV infects T cells in EBV-associated diseases, including hemophagocytic lymphohistiocytosis, T cell lymphoma or leukemia, and chronic active EBV infection. Despite the growing evidence of the development of T or NK cell lymphoproliferative disease in patients with chronic active EBV infection [12, 13], the mechanism of infection remains elusive. It is not clear whether activated T cells in these patients take a salient part in the resistance to EBV, or otherwise, in the disease progression. A Th1 or Th2 paradigm has been established in murine models for a decade [14]. The biological role of polarized T cells has been investigated in various types of infection. Nevertheless, this distinction is less clear in humans, and the cytokine secretion profile is difficult to approach in clinical infectious diseases. Recently, real-time polymerase chain reaction (PCR) has enabled us to complete the quantitative analysis of gene expression [15, 16]. To date, there are a few reports about cytokine gene expressions in EBV-associated lymphoproliferative disease or lymphoma but not in chronic active EBV infection. In this study, quantitative real-time PCR and cell sorting were used to verify the expression of EBV DNA and cytokines in activated T cells obtained from patients with chronic active EBV infection. Patients, Materials, and Methods Patients. Six Japanese children (4 boys and 2 girls) who fulfilled the diagnostic criteria of chronic active EBV infection [7, 8] were enrolled for the study. Clinical characteristics are shown in table 1. There were neither consanguineous marriages nor immuno- To identify the role of T cells in chronic active Epstein-Barr virus (EBV) infection, EBV and cytokine gene expression was quantified by use of real-time polymerase chain reaction (PCR) among 6 patients who fulfilled the diagnostic criteria for chronic active EBV infection. Four of these patients showed clonal expansion of EBV-infected T cells. Quantitative PCR for EBV DNA in peripheral blood of patients with symptomatic chronic active EBV infection showed higher copy numbers of virus (mean, 1.45 3 10 5 copies/mL) than were seen in blood from patients with infectious mononucleosis (3.08 3 10 3 copies/mL) or with EBV-associated hemophagocytosis (2.95 3 10 4 copies/mL). Fractionated CD3+ HLA-DR+ cells from patients with chronic active EBV infection contained higher copy numbers than did CD3+ HLA-DR2 cells. Quantitative PCR for cytokines revealed that interferon-g, interleukin (IL)–2, IL-10, and transforming growth factor–b genes were expressed at higher levels in HLA-DR+ than in HLA-DR2 T cells. These results suggest that activated T cells in chronic active EBV infection expressed high levels of EBV DNA and both Th1 and Th2 cytokines. EBV-infected T cells may contribute to the unbalanced cytokine profiles of chronic mononucleosis. 2 Ohga et al. Table 1. Characteristics of patients with chronic active Epstein-Barr virus (EBV) infection. Patient 1 2 3 4 5 6 Age, years Sex Age at onset, years Clinical findings Coronary lesion Chorioretinitis CNS disease a EBV antibodies Viral capsid antigen IgG IgA IgM Early antigen IgG IgA EBV nuclear antigen CD31 HLA-DR1, b % of cells c CD41 : CD81 EBV clonality T cell antigen receptor rearrangement 10 M 6 16 F 9 10 F 8 15 M 12 3 M 2 1 M 1 Yes No No Yes Yes Yes No No No No No No No No No No No No 1280 10 !10 10,240 320 !10 2560 20 !10 5120 160 !10 1280 !10 !10 20,480 20 20 1280 10 10 640 20 10 320 10 20 1280 40 40 80 !10 160 1280 ND 20 60 4.4 Mono 19 1.5 Mono 11 0.6 Oligo 20 0.7 Mono 17 1.2 ND 16 3.5 Poly R R G G ND ND NOTE. CNS, central nervous system; F, female; G, germline band; M, male; Mono, monoclonal band; ND, not determined; Oligo, oligoclonal band; Poly, polyclonal smear; R, rearrangement band. a Cutoff, each !10. b Normal mean, 7.5% (range, 4.0%–9.0%). c Normal mean, 1.43 (range, 0.8–2.9). hematologic disorders in their relatives. No patients had mosquito allergy. They showed 110% HLA-DR1 CD31 cells but no increase in CD161 or CD561 cells in peripheral blood. Southern blot analysis probed with EBV terminal repeats revealed 1 band in patients 1, 2, and 4 and 3 bands in patient 3. T cell antigen receptor Cb/ Jb2 gene rearrangements were defined in patients 1 and 2 but not in patients 3 and 4. Patients 1 and 2 had severe disease [9]. At the time of the patients’ examination, 15 years had passed since the onset of their illness. Patient 1 presented with fever and splenomegaly at 6 years of age. Coronary artery aneurysm and or (...truncated)