Restricted Diversification of T-Cells in Chronic ActiveEpstein-Barr Virus Infection: Potential Inclination to

T-Lymphoproliferative Disease

Shouichi Ohga, 1* Nobuhiro Kimura, 2 Hidetoshi Takada, 1 Mituyuki Nagano, 2

Kohichi Ohshima, 3 Akihiko Nomura, 1,4 Kenji Muraoka, 4 Hiromichi Take, 4 Shunji Yamamori, 5

and Toshiro Hara 1

1Department of Pediatrics, Faculty of Medicine, Kyushu University, Fukuoka, Japan2The First Department of Internal Medicine, School of Medicine, Fukuoka University, Fukuoka, Japan

3The First Department of Pathology, School of Medicine, Fukuoka University, Fukuoka, Japan4Division of Pediatrics, Kagoshima Municipal Hospital, Kagoshima, Japan

5Division of Gene Analysis, Mitsubishi Bio-Chemical Laboratories, Inc., Tokyo, Japan

To assess the abnormal T-cell expansion in chronic active Epstein-Barr virus infection(CAEBV), T-cell antigen receptor (TCR) repertoire was analyzed in four patients with thedisease. All fulfilled the diagnostic criteria of CAEBV, presenting with fever, hepato-splenomegaly, cytopenia, abnormal high titers of anti EBV-antibodies, and positive EBVgenome of unknown cause. Southern blotting probed with EBV-terminal repeats and TCRCb gene indicated clonal expansion of the infected cells in 3 and 2 patients, respectively.The number of CD4 + HLA-DR+ cells appreciably increased in patients 1 (59%) and 2 (24%),who had a coronary aneurysm and central nervous system involvement, respectively.TCR gene expression examined by the inverse polymerase chain reaction methods re-vealed that V b gene usages were preferential in all patients (V b7 and Vb12: patient 1,Vb4: patient 2, V b13: patients 3 and 4), compared with those in healthy controls. V a18gene expression was remarkably high in patients 1 and 2. Moreover, J b gene expressionwas skewing in the reigning V b clones in all patients. V b4-Jb1.5 and Vb13-Jb1.5 geneswere clonally expressed in patients 2 and 4, respectively. These results suggest thatCAEBV is associated with the restricted diversity of T-cells, which may stem from thesustained expansion of oligoclonal T-cells possibly driven by conventional viral anti-gens, but not, superantigens. Although the study is limited by the small number ofpatients, the unbalanced T-cell repertoire might contribute to the evolution of T-lymphoproliferative disease, otherwise, imply the innate defective immunity to EBV inCAEBV patients. Am. J. Hematol. 61:26–33, 1999. © 1999 Wiley-Liss, Inc.

Key words: CAEBV; EBV genome; LPD; TCR repertoire

INTRODUCTION

Primary infection of Epstein-Barr virus (EBV) leads tosubclinical or acute infectious mononucleosis (IM) insusceptible immunocompetent subjects. EBV is incrimi-nated as a causative agent in an array of malignanciesincluding Burkitt’s lymphoma, Hodgkin’s or non-Hodgkin’s lymphoma, nasal T-cell lymphoma, pyotho-rax-associated lymphoma and nasopharyngeal, thymic,or gastric carcinoma [1–6]. Primary and secondary im-munodeficiencies occasionally elicit the persistent reac-tivation of EBV and the subsequent development ofEBV-associated lymphoproliferative disease (LPD),most of which originate from the outgrowth of EBV-

infected B lymphocytes [7–10]. The clinical entity ofchronic active EBV infection (CAEBV) has been estab-lished as a primary persistent mononucleosis character-

Contract grant sponsor: Ministry of Health and Welfare for PrimaryImmunodeficiency Diseases; Contract grant sponsor: Ministry of Edu-cation, Science and Culture of Japan.

*Correspondence to: Shouichi Ohga, M.D., Department of Pediatrics,Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku,Fukuoka 812-8582, Japan. E-mail: ohgas@mailserver.med.kyushu-u.ac.jp

Received for publication 27 July 1998; Accepted 6 January 1999

American Journal of Hematology 61:26–33 (1999)

© 1999 Wiley-Liss, Inc.

ized by fever, cytopenia, hepatosplenomegaly alongwith the clonal proliferation of EBV [11–14]. This dis-ease is supposed to render the defective T-cell immunityto EBV and develop the lymphoid malignancy [15,16].There has been convincing evidence that EBV can infectand proliferate T-cells and natural killer (NK) cells aswell as B-cells [17–22]. It may be associated with theevolution of clonal lymphoproliferation and/or lympho-mas of T/NK-cells; however, the pathogenesis has notbeen elucidated.

In this study, we investigated the T-cell repertoire inCAEBV patients by using the novel inverse polymerasechain reaction (PCR) methods [23], which could allow usto perform a quantitative analysis on the usages of T-cellantigen receptor (TCR) variable (V)/joining (J) regiongenes in lymphocytes. The possible association betweenthe aberrant T-cell expansion and the clonal evolution inCAEBV is then discussed.

PATIENTS and METHODSClinical Profiles of Patients

Clinical characteristics of four Japanese children (3males and 1 female) with CAEBV are shown in Table I.Each patient fulfilled the diagnostic criteria of the disease[11,12]. There was no consanguineous marriage, or im-munohematologic disorders in their families. None ofthem had mosquito allergy [22]. Patient 1, a 6-year-oldboy first presented with splenomegaly and periodic highfever [24]. This patient deteriorated with the develop-ment of coronary artery aneurysm, which subsided byinterferon (IFN)-a therapy. Peripheral blood was ob-tained for this study before the administration of IFN-a.Patient 2 was diagnosed with CAEBV at 14 months ofage. Various noncytotoxic agents could not confer theprolonged resolution. At 8 years of age, he was splenec-tomized for the disease progression accompanied by AVblock and retinopathy. Following 2 years of clinical re-mission, central nervous system (CNS)-LPD arose. Brainbiopsy disclosed the infiltration of CD4+, CD45RO+, andEBV-encoded RNA (EBER) 1+ T-cells. Blood samplingwas performed at the time of the diagnosis of CNS-LPD.Patients 3 and 4 were diagnosed with CAEBV at 14 and12 years of age, respectively. They showed hepatitis andsplenomegaly, but required no intensive treatment formore than 2 years after the onset of disease.

Surface Marker Analysis

Peripheral blood was obtained from four CAEBV pa-tients. Informed consent was obtained from each patientand/or their guardians for this study. Flow cytometricanalysis was performed using an EPICS XL (Coulter,Miami, FL) as described previously [25]. The forwardlight scatter gate was set to analyze viable lymphocytesand exclude background artifacts. Multi-color staining

was carried out using fluorescein isothiocyanate (FITC)-,phycoerythrin (PE)-, or phycoerythrin-cyanin 5.1(PECy5)-conjugated monoclonal antibodies (Abs) ofTCR1ab (Becton Dickinson, San Jose, CA), CD3, CD4,HLA-DR (Becton Dickinson; Immunotech Coulter, Mi-ami, FL), CD8, CD19, CD56 (Immunotech Coulter).

Detection of EBV Genome

High molecular DNA was extracted from peripheralmononuclear cells (PMC), bone marrow (BM), or spleencells. Dot blotting to detect the EBV genome was per-formed based on the conventional PCR protocol, usingthe EBV primer specific for a 240 base pair (bp) regionwithin the BamHI-K region containing the internal re-peated (IR) sequences of the EBV genome as describedpreviously [19]. In this PCR protocol, DNA extractedfrom a cell line (Raji) or the PMC of a patient with acuteIM shows positive, and DNA from the PMC of healthysubjects who had no antibodies to EBNA shows negativeEBV genome.

TABLE I. Characteristics of Patients With Chronic ActiveEpstein-Barr Virus Infection*

Patients 1 2 3 4

Age (years) 8 11 16 14Age of onset (years) 6 1y 2m 9 12Sex M M F MClinical findings

Fever Yes Yes Yes YesCytopenia Yes Yes Yes YesHepatosplenomegaly Yes Yes Yes YesArrhythmia No Yes No NoCoronary lesiona Yes No No NoOcular involvement No Yes No NoCNS lesiona No Yes No No

Laboratory findingsImpaired PHA response No Yes No NoDepressed NK activity Yes Yes No No

VCA-IgG 1,280 10,240 10,240 5,120IgA 10 160 320 160IgM <10 <10 <10 <10

EADR-IgG 1,280 640 640 1,280IgA 10 <10 20 40

EBNA 10 160 10 40CD3+HLA-DR+ (%) 60 49 19 20CD4+CD8− (%) 59 24 14 4CD4−CD8+T (%) 6 14 7 16CD4/CD8 4.4 1.9 1.5 0.7EBV clonalityb Mono Poly Mono MonoTCR rearrangementb R G R GIg rearrangementb G NT G G

*M, male; F, female; EBV, Epstein-Barr virus; TCR, T-cell antigen recep-tor; Ig, immunoglobulin; Mono, monoclonal band; Poly, polyclonal smear;R, rearrangement band; G, germline band; NT, not tested.aBlood sampling was performed before the development of coronary lesionin patient 1, and at the diagnosis of central nervous sytem (CNS)-lymphoproliferative disease in patient 2.bEBV clonality and TCR/Ig gene rearrangement were assessed by Southernblotting.

TCR Repertoire in CAEBV 27

Southern Blot Analysis Probed WithEBV-Terminal Repeats or TCR/Ig Genes

Southern blotting was performed as described previ-ously [19]. Briefly, 5mg of high molecular weight DNAwere digested withEcoRI, BamHI, HindIII, and/orKpnI.The digested DNA was electrophoresed on 0.9% agarosegel and transferred to Byodine-B, which was hybridizedwith a 32P-labeled probe. Clonality of lymphocytes wasassessed using the probes of a 5.2-kbBamHI-EcoRI frag-ment containing the tandem terminal repeated (TR) se-quence of the EBV genome, and/or the TCR/immunoglobulin (Ig) gene probes of Cb1, Jb1, Jb2, Cg,IgH, k, andl.

Inverse PCR Method for Analyzing TCR V a,Vb,and J b Gene Usages

The newly devised inverse PCR method was appliedto analyze the TCR repertoire [23]. Briefly, oligo(dT)-primed double stranded cDNA was synthesized from 1mg of total RNA using reverse transcriptase, RNase H,Escherichia coli(E. coli) DNA polymerase I, andE. coliDNA ligase, followed by the incubation with T4 DNApolymerase for blunt-end formation. Following the con-struction of circular double stranded cDNA by T4 DNAligase, PCR was performed separately using two constantregion primers of Ca or Cb that are in opposite direc-tions: Ca (forward), 58-GGG TCG ACG ACC TCA TGTCTA GCA CAG T-38; and Ca (inverse) , 58-GCA TGCGGC CGC CCT GCT ATG CTG TGT GTC T-38; or Cb(forward), 58-GGG TCG ACC TGT GCA CCT CCTTCC CAT T-38, and Cb (inverse), 58-GCA TGC GGCCGC ATG GCC ATG GTC AAG AGA-38. After 30cycles of PCR (denaturation at 95°C for 30 sec, anneal-ing at 62°C for 30 sec, and extension at 72°C for 1 min),the amplified PCR mixture was electrophoresed on 1.5%low-melting-point agarose, and then the cDNA frag-ments expected sizes between 600 bp and 950 bp wereextracted to be used as a probe. The cDNA fragmentswere labeled witha-32P-dCTP and hybridized to the fil-ters, on which 200 ng of each Vb fragment (Vb1≈Vb20)or Va fragment (Va1≈Va18, Va21, Va24) were dotted.Each V-specific fragment size ranging from 280 bp to350 bp was prepared from the series of HBVT/HBVP orHAVT/HAVP plasmids originated from thymus or pe-ripheral T-cell [26]. Since this PCR protocol employstwo opposite directional C region gene primers to behybridized with each V region gene, the spot intensityrepresents the V gene usages within the equally ampli-fied TCR clones. Using the densitometry, the quantita-tive assessment of V gene usage was made from the ratio(%) of the total amounts of hybridized products on eachV family gene. When a certain Vb gene was preferen-tially used, Jb gene usages in the corresponding Vbclone were also examined. RT-PCR amplification of the

RNA was performed using the sense Vb gene primer anda total of 13 sets of antisense Jb primers. The antisenseJb primers were as follows: Jb1.1, 58-AAC TGT GAGTCT GGT GCC TTG-38; Jb1.2, 58-AAC GGT TAACCT GGT CCC CGA-38; Jb1.3, AAC AGT GAG CCAACT TCC CTC-38; Jb1.4, 58-GAC AGA GAG CTGGGT TCC ACT-58; Jb1.5, 58-GAT GGA GAG TCGAGT CCC ATC-38; Jb1.6, 58-CAC AGT GAG CCTGGT CCC ATT-38; Jb2.1, 58-CAC GGT GAG CCGTGT CCC TGG-38, Jb2.2, 58-TAC GGT CAG CCTAGA GCC TTC-38; Jb2.3, 58-CAC TGT CAG CCGGGT GCC TGG -58; Jb2.4, 58-CAC TGA GAG CCGGGT CCC GGC-38; Jb2.5, 58-CAC CAG GAG CCGCGT GCC TGG-38; Jb2.6, 58-CAC GGT CAG CCTGCT GCC GGC-38; Jb2.7, 58-GAC CGT GAG CCTGGT GCC CGG -38. The sense Vb primers were asfollows: Vb4, 58-TGA GGC CAC ATA TGA GAGTGG-38; Vb7, 58-GCT TCT CAC CTG AAT GCCCCA-38; Vb12, 58-ACT CTG AGA TGT CAC CAGACT-38; Vb13, 58-ACA CTG CAG TGT GCC CAGGAT-38.

RESULTS

Anti EBV-Abs, Surface Marker, and EBV Genome

The results of anti EBV-Ab titers, surface markeranalysis, and the assessments of EBV genomes and TCR/Ig genes are shown in Table I. All patients showed ab-normal high titers of EB VCA-IgG ($1280), EADR-IgG($640) and the detection of VCA-IgA ($10), and nega-tive VCA-IgM and positive EBNA. CD3+HLA-DR+

cells increased, of which CD4+ phenotype predominatedin patients 1 and 2. On the other hand, patients 3 and 4showed lower number of activated T-cells and lowerCD4/CD8 ratio than patients 1 and 2.

EBV genome was assessed by the dot blotting PCRmethod probed with EBV-IR sequences, Southern blot-ting probed with EB-TR sequences, and/or in situ hy-bridization (ISH) using EBV-encoded RNA (EBER)1.EBV genomes were detected in all patients by the PCR-products method (data not shown). Southern blotting us-ing TR sequence indicated the monoclonal nature (singleband) of the EBV in patients 1, 3, and 4 (Table I).EBER1-ISH exhibited the high frequency of EBER+

cells in tissue specimens in patients 2 (brain, spleen) and4 (lymph node) (data not shown). When TCR or Ig generearrangements were assessed by Southern blotting ofDNA samples obtained from the PMC using TCR Cb, g,Jb1, 2 or JH,k, l gene probes, the rearranged bands ofCb, g, and Jb2 genes were determined in patient 1, andthose of Cb gene were detected in patient 3 (Fig. 1). Thegerm line configuration was found in Ig genes studied.

28 Ohga et al.

TCR Va and Vb Gene Usages inPeripheral Lymphocytes

TCR Va/bgene usages of lymphocytes obtained fromthese patients were examined by the inverse PCR meth-ods (Fig. 2). In patient 1, Va5 (49%) and Va18 (35%)genes occupied more than 80% of total TCR Va geneexpression, and Vb7 (29%) and Vb12 (23%) genes werepreferentially expressed. In patient 2, Va1 (22%) andVa18 (29%) genes attained to the half of total expres-sion, and Vb4 (32%) and Vb13 (13%) genes were fre-quently expressed. In patient 3, no Va gene exceeded15% of total Va gene expression, however, Vb13 genewas remarkable at 28% of Vb gene expression. In patient4, Va3 (22%) and Vb13 (27%) genes were selectivelyused. Healthy controls did not show any specific clonewhich exceeded more than 15% of total Va or Vb geneexpression.

Jb Gene Expression in the Dominant V b Clones

To further investigate the clonality of T-cells bearingthe specific Vb gene, the Jb gene usages of PCR prod-ucts containing the specific Vb gene were analyzed (Fig.3). In patient 1, Jb2.1 (25%) and Jb2.7 (25%) fragmentswere prominent in Jb-PCR products for Vb7 gene. Jb2.7(35%) was also frequently expressed in Vb12 clones. Inpatient 2, Jb1.5 (81%) was exclusively used for Vb4-PCR products. In patient 3, Jb1.4 was expressed to 44%of the total expression of Vb13 gene. In patient 4, Jb1.5occupied 78% of Vb13 gene expressed. In summary, theskewing usages of Vb gene and the preferential recom-bination of specific Jb gene were outstanding in allCAEBV patients. Vb4-Jb1.5 and Vb13-Jb1.5 geneswere clonally expressed in patients 2 and 4, respectively.The usage of the Va18 gene was predominantly ob-

Fig. 1. TCR gene rearrangement patterns in patients 1 (upper) and 3 (lower) with CAEBV infection. Southern blot analysisof cellular DNA obtained from peripheral blood was performed with TCR C b, g, Jb2 gene probes as described previously[24]. E, EcoRI; B, BamHI; H, Hind III; K, Kpn I; G, germ line band; R, rearrangement band.

TCR Repertoire in CAEBV 29

served in patients 1 and 2, while the Vb13 gene wasselectively expressed in patients 3 and 4 (Table II).

DISCUSSION

The notable finding of the present study was the re-stricted diversity of TCR Vb genes with the preferentialJb gene expression in all CAEBV patients. TCR Va geneusages were also restricted in 3 of 4 patients. This skew-ing TCR pattern was conspicuous compared with thevariable diversification of healthy human TCR Va, Vb,and Jb repertoire described previously [27], and that ofour controls (Fig. 2). There was no report on the T-cellrepertoire in persistent reactivation of EBV. Silins et al.[28] revealed the selection of a diverse TCR repertoire inresponse to anti-EBV encoded transactivator protein byCD8+ CTL, and suggested that the biological role inmaintaining a balanced viral load throughout EBV per-

sistence. Smith et al. [29] reported the increased expres-sion of Vb6.1-3 and Vb7 T-cells in acute IM, and pro-posed the superantigen response. Sutkowski et al. [30]have revealed the selective activation of human Vb13+

T-cells in response to EBV-infected cells, suggesting therole of EBV-associated superantigen in EBV infection.They further speculated that the symptoms of IM mightbe explained by the cytokine production and subsequentnonspecific T-cell activation via the superantigen-induced Vb13+ T-cells, and that the slower and weakersuperantigen response in children was balanced by theinduction of EBV-specific CTL to prevent clinical mani-festation. In fact, the preferential expansion of Vb13gene was observed in patients 3 and 4; however, the Jbgene usages indicated oligo- or mono-clonal expansionof Vb13+ cells (Fig. 3). Furthermore, Jb2.7 gene expres-sion in Vb7 and Vb12 clones in patient 1 well reflectedthe Cb and Jb gene rearrangements assessed by Southern

Fig. 2. TCR Va or Vb gene usages of peripheral lympho-cytes by the inverse PCR method [23]. The double strandedcDNA was synthesized from total RNA obtained from pe-ripheral blood of four patients. Following the circularizationof the blunt-ended cDNA, PCR was separately performedusing two constant region gene primers in opposite direc-tions: C a (forward and inverse) or C b (forward and inverse).

The PCR mixture was electrophoresed on agarose, and theappropriate-sized cDNA fragments were extracted. The frag-ments were labeled with a-32P-dCTP and hybridized to theVb (from V b1 to Vb20) or Va fragments (from V a1 to Va18,Va21, Va24) dotted filters. These bar graphs depict thequantitative assessment of the V gene usages using densi-tometry.

30 Ohga et al.

blotting (Fig. 1). Callen et al. [31] have revealed recentlythat large monoclonal or oligoclonal populations ofCD8+ T-cells expanded in acute phase of IM, and disap-peared in convalescent phase. Sequence analysis ofCDR3 and its corresponding Jb gene selection stronglyindicated that these populations had been driven by viralantigens. In this context, our data support that the sus-tained expansion of T-cells was clonally driven by con-ventional viral antigens rather than superantigens. Hu-man TCR V-segment frequencies in peripheral blood are

influenced profoundly by the HLA complex [32]. Argaetet al. [33] described the dominant selection of an invari-ant TCR from HLA-B8-restricted CD8+ cytotoxic T-cellclones in response to persistent infection by EBV. Thedisparate TCR diversity in each patient may partly ac-count for the differences in the HLA complex and/or theproportion of CD8+ T-cells.

Imai et al. [17] established four T-cell lines infectedwith EBV, one of which expressed CD4+ CD8− CD45+

phenotype accompanied by clonal TCRb and g generearrangements. Both rearrangements of TCRb and ggenes were also determined in patient 1. Ishihara et al.[22] described clonal lymphoproliferation in CAEBV pa-tients, two of whom showed the peripheral increase ofCD4+ T-cells. In patients 1 and 2, the increase of acti-vated CD4+ CD8− T-cells correlated with the diseaseprogression. Histopathological analysis of the brain le-sion in patient 2 revealed that the major infiltration wascomposed of EBV-infected activated CD4+ T-cells (datanot shown). Paterson et al. [34,35] indicated that humanimmature thymocyte was activated after EBV infection

Fig. 3. J b gene usages in the predominated V b clones. When a certain V b gene was preferentially used, RT-PCR ampli-fication was carried out using the sense V b gene primer and a total of 13 sets of antisense J b primers. These bar-graphsillustrate the ratio of each hybridized intensity of J b gene products to the total amounts of hybridization. Each figure belowthe graph shows the real intensity of hybridization.

TABLE II. T-cell Repertoire in Patients With Chronic ActiveEpstein-Barr Virus Infection

Patient ComplicationTCR gene

rearrangementa

TCR V or J gene usagesb

Va Vb Vb/Jb

1 Severe Cb,Jb2,Cg 5,18 7,12 7/2.7, 12/2.72 Severe No 1,18 4 4/1.53 Nonsevere Cb Variable 13 13/1.44 Nonsevere No 3 13 13/1.5

aSouthern blotting.bInverse PCR method.

TCR Repertoire in CAEBV 31

and its consequences at an early stage of differentiationmight lead to failure of normal T-cell repertoire devel-opment. Taken together, the clonal expansion of EBV-infected T-cells may affect the derangement of T-celldiversity and contribute to lymphoproliferation inCAEBV patients.

From another point of view, the reduced fractions ofeach Va, Vb T-cell may be of much more importancethan the expanded subpopulations. The engrafted hema-topoietic cells can occasionally express a restrictedclonal diversity of T-cells in severe combined immuno-deficiency [36]. The clonal expansion and exhaustion ofT-cells might also occur in patients with human immu-nodeficiency virus infection. Patients with X-linked lym-phoproliferative disease (XLP) succeed to the defectivecellular immunity to EBV and develop LPD/lymphomasas a consequence. In the present study, clonal T-cell pro-liferation was not always associated with the clinical se-verity and the increased number of activated T cells(Table II). It may raise speculation that CAEBV-T cellscarry an indigenous defect to organize the balanced T-cell repertoire during persistent EBV infection. Furtherfractionation analyses on EBV-infected T-cells are re-quired not only for understanding the pathogenesis ofCAEBV, but also for characterizing the clinical entity ofprimary T-cell deficiency disease triggered by EBV in-fection.

ACKNOWLEDGMENTS

We are grateful to Takaaki Kanamaru, MT (Morphol-ogy Core, Faculty of Medicine, Kyushu University,Fukuoka, Japan) for technical assistance, and Brian T.Quinn (Associate Professor, Kyushu University,Fukuoka, Japan) for reviewing this manuscript. Thiswork was supported in part by a grant from the Ministryof Health and Welfare for Primary ImmunodeficiencyDiseases, and by a grant-in-aid for the encouragement ofyoung scientists from the Ministry of Education, Scienceand Culture of Japan.

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