Microbiol.

Immunol.

Vol. 36 (5), 479-494, 1992

Induced

CD25 Expression

Cell Line Transfected Virus Nuclear Shizuko

in a Human

B-Lymphoma

with the Epstein-Barr Antigen 2 Gene

HARADA,4 and

Kazuo

YANAGI*

Department of Viologyand Rickettsialogy,National Institute of Health, Shinagawa-ku,

Tokyo 141, Japan

(Accepted for publication, February 24, 1992)

Abstract Two EBV-negative human B-lymphoma cell lines, BJAB and DG75, were transfected with an Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) gene, which plays a critical role in the EBV-induced immortalization of primary B lymphocytes. Furthermore, DG75 cells were co-transfected with the EBNA-2 gene and a latent membrane protein (LMP) gene. Expression of eight surface antigens on the resultant EBNA-2-expressing cell clones was analyzed by flowcytometry. None of the EBNA-2-expressing cell clones derived from BJAB and DG75 showed a significant increase in the expression of cell surface marker CD23, of which enhancement by EBNA-2 in a different EBV-negative human B cell line, Louckes, was previously reported. Expression of CD25 (IL-2R/Tac) on cell surface, however, was induced in two of six DG75-derived cell clones. One of the two CD25-induced cell clones was expressing EBNA-2 only, and the other was co-expressing EBNA-2 and LMP. The results suggest that EBNA-2 has a potential to up-regulate CD25 independently of CD23 on human B cells.

Epstein-Barr virus (EBV) has been considered to be associated with Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (12). Infection of normal human resting B cells with EBV in vitro immortalizes the cells and generates lymphoblastoid cell lines (LCLs) which carry multiple copies of the latent EBV genome (13). In LCL cells, at least eight latent viral proteins are expressed, consisting of six nuclear antigens (EBNAs), i.e. EBNA-1, -2, -3A (-3), -3B (-4), -3C (-6), -LP (-5), and two latent membrane antigens, LMP1 and LMP2 (terminal protein) (11, 18). Circumstantial evidence has suggested that EBNA-2 is an essential gene for the immortalization of B cells by EBV. The EBV strain P3HR-1, of which genome lacks a segment containing the EBNA-2 gene (27, 34), is incapable of immortalizing resting B cells (22, 25). The two types of EBV, EBV-1 and EBV-2, which were differentiated initially based on antigenicity of EBNA-2, differ from each other in the characteristics of growth-transformation of B lymphocytes (6, 33, 35). Recently, 〓Present

affiliation:

Department

of Medical

Entomology. 479

480

S. HARADA

AND K.

YANAGI

more direct evidence for the indispensable role of EBNA-2 in the B cell immortalization has been reported by Hammerschmidt and Sugden (17) and Cohen et al (7), who demonstrated that EBNA-2 but not EBNA-5 (EBNA-LP), which is also located in the deleted region of the P3HR-1 genome, complemented P3HR-1 in the B cell immortalization. It was also reported that the EBNA-2 expression in Rat-1 cell line altered its cell growth properties such as serum requirement (10). Moreover. in an EBV-negative BL cell line, Louckes, EBNA-2 induced formation of large cell clumps and augmented expression of a particular B cell activation antigen CD23 (47), of which expression is known to increase on the surface of B cells infected with EBV (42). We attempted to analyze directly whether EBNA-2 potentiates the immortalization or growth-transformation of human primary B lymphocytes in the initial stage of this study. However, it was very difficult to express the EBNA-2 gene sufficiently in human primary B lymphocytes which were prepared from cord blood, because the efficiency of gene transfer into the primary B cells was lower than 10-6 even by the method of electroporation. We, therfore, turned to human B cell lines as recipient cells for the expression of the EBNA-2 gene to analyze its effect(s) on growth-related properties of already immortalized human B cells, in particular on expression of their cell surface markers. In this study, we constructed an EBNA-2-expressing plasmid and an LMYexpressing plasmid, and made a set of cell clones which expressed EBNA-2 by transfection with the plasmid into two human B-lymphorr a cell lines, BJAB and DG75. In the analyses of the generated cells, induction of CD25 (IL-2R/Tac) was found in two DG75-derived cell clones expressing EBNA-2, one of which was cotransfected with the LMP gene. MATERIALS

AND

METHODS

Constructionof recombinantplasmids. Plasmid pKCRE2 (Fig. 1A) was constructed by subcloning the 1.8 kb AccII-Dral fragment [nucleotide number (nt. no.) 48474 to 50305, according to Baer et al (4)] of the EBV B95-8 strain DNA into the unique HindIII site of the vector plasmid pKCRH2 (26) (given by Y. Morimoto, Mitsubishi Chemical Co., Tokyo). The -AccII-DraI fragment was excised from P5 cosmid DNA which carries a contiguous BamHI C, W, Y, H, and F fragments of the EBV genomic DNA. The cosmid P5 was provided by B. E. Griffin (16). Plasmid pSVLMPneo (Fig. 1B) was constructed by subcloning the 3.7 kb SstII fragment, which corresponds to nt. no. 166451-170152, derived from the cosmid 996-20 (given by G.W. Bornkamm) which has the EcoRI fragment containing the covalently linked termini of the circularized genome of the EBV strain M-ABA (31) and the 3.6 kb Accl-EcoRI fragment from pSV2neo (38) into the HindIll and EcoRI sites of pSV2gpt (29), respectively. The vector plasmids pSV2gpt and pSV2neo were gifts of Y. Morimoto and K. Oda (Tokyo Science Univ.), respectively. Plasmid pC43 (Fig.1C) was constructed by ligating the BamHI-PvuII subfragment (nt. no. 107565110176) from BamHI K fragment of B95-8 (given by E. Kieff) (9), into the EcoRI

CD25

INDUCTION

IN B CELLS

EXPRESSING

EBNA-2

481

site of pSV2gpt with the SV40 origin-promoter element excised from pSV2 vector (28). The orientations of the inserts relative to the SV40 early promoter elements were determined by restriction endonuclease digestion of the recombinant plasmid DNAs. Cells. BJAB (23) and DG75 (5), which were provided by G. Klein, and Louckes (a gift of A.B. Rickinson), and Ramos (obtained from ATCC: American Type Culture Collection) are all EBV genome-negative BL cell lines. B95-8 is an EBVproducing marmoset B cell line (24). Raji is an EBV-positive BL cell line, and was obtained from ATCC through the Japanese Cancer Research Resources Bank. LCL-C4 was generated in our laboratory by infection of cord blood B lymphocytes with EBV (B95-8). These lymphocyte cell lines were grown in RPMI 1640 medium (Nissui, Tokyo) supplemented with 10% heat-inactivated fetal calf serum (Flow Laboratories), 100 mg/ml kanamycin (Meiji Seika, Tokyo) and 1.25 mg/ml amphotericin B (Boehringer Mannheim). Transfection and isolation of cell clones. Plasmid DNAs were introduced into their target lymphocytes by electroporation with a Gene Pulser (Bio-Rad) or an electroporation apparatus made in collaboration with Olympus Co. (Tokyo). The cells were suspended at 5 x 107 per ml in 300 ,u1of serum-free RPMI 1640 medium containing 10 pg plasmid DNA in a 4-mm-path cuvette and exposed, in most cases, to an electric pulse at 600 volts and 25 ILK The plasmid pKCRE2 was co-transfected into BJAB or DG75 cells with pSV2gpt at a molar ratio of 10 : 1 to generate EBNA-2expressing and mycophenolic acid (MPA)-resistant cells. To obtain cell clones expressing both EBNA-2 and LMP, the two plasmids pKCRE2 and pSVLMPneo were co-transfected into DG75 cells. Sixteen hours after the electroporation, cells were distributed in flat-bottom 96-well plates (Corning) at the concentrations of 1,000, 2,500, or 5,000 cells per well in medium containing MPA (Sigma) at 1 pg/ml and xanthine (Sigma) at 200 ,ug/ml, or in medium containing G418 (GIBCO) at 3 mg/ml when pSVLMPneo was transfected. The cell cultures were fed once a week with fresh medium containing the drugs. The obtained drug-resistant cell clones were transferred from the wells each of which contained only a single cell clone into larger culture dishes. Immunofluorescence analysis. Lymphocytes were mounted on microscopic slides, fixed by acetone–methanol (1 : 1) at -20 C, then processed for anti-complement immunofluorescence (ACIF) analyses (32). Expression of EBNA-1 and EBNA-2 was analyzed with human serum, KM, which was EBNA-positive. Immunoblot analysis. Selected drug-resistant cells were lysed (5 x 106 cells in 0.1 ml) in SDS sample buffer (20), followed by sonication for 10 sec and centrifugation at 15,000 rpm for 5 min. The supernatants were boiled for 5 min and electrophoresed in SDS-polyacrylamide gel (20). The separated proteins were transferred electrically to a nitrocellulose filter sheet as described by Towbin et al (43). Immediately after transfer, the filter sheets were blocked with skim milk, incubated with the forty-fold diluted KM serum in TBS (20 mm Tris-Cl pH 7.5, 500 mm NaCl) containing 0.1% skim milk for 14 hr at 4 C. The filters were treated with antihuman IgG monoclonal antibody (ICN ImmunoBiologicals, Lisle, Ill., U.S.A.)

482

S. HARADA

AND K. YANAGI

and incubated with biotinylated anti-mouse immunoglobulins (Dakopatts, Grostrup, Denmark) and then with peroxidase-conjugated avidin (Dakopatts). The filters were washed 3 times with TBS containing 0.005% Tween 20 at each step. Finally, the sheets were developed in TBS containing hydrogen-peroxide and 4-chloro-lnaphtol (BIO-RAD). Flowcytometryanalysis. Approximately 1 •~106 viable cells were treated with a panel of FITC- or phycoerythrin-conjugated monoclonal antibodies on ice for 30 min in a dark room. The FITC-conjugated monoclonal antibodies were as follows: anti-HLA-DQ, anti-HLA-DR, anti-CALLA, anti-IL-2 receptor (Becton Dickinson, Cat. no. 7453, 7363, 7503, 7643, respectively), B 1, B4 (Coulter Immunology), H107 (Nichirei, Tokyo). Phycoerythrin-conjugated B2 was from Coulter Immunology. After washing with Hank's salt solution, the antibodylabeled cells were resuspended in 400 ill of Hank's salt solution, then their immunofluorescence was analyzed with a FACS 440 cell sorter (Becton Dickinson). The specificity of the panel of monoclonal antibody is shown in Table 1. The secondary antibody reactions were done with FITC-conjugated anti-mouse IgG goat antibody (Tago), when MAb25 or S12 (described below) was used as the primary antibody. MAb25 was used to specify CD23 and was a gift of J. Yodoi (Kyoto University). The anti-LMP monoclonal antibody S12 ascites was a gift of D.A. Thorley-Lawson, and used for flowcytometry analyses according to his cell-fixation method (21). Chloramphenicol acetyltransferase assay. Analyses of chloramphenicol acetyltransferase (CAT) activity were performed according to the protocol given by B.H. Howard (15). The EBNA-2-expressing cells were transfected by electroporation with pSV2CAT (15), pCHL4 (36), pWEN-CAT (36), and pA 1OCAT as a control. BJAB cells were co-transfected with pKCRE2 or the vector pKCRH2 in combination with pSV2CAT, pSRaCAT (40), pCHL4, or pWEN-CAT by electroporation. Both pSV2 CAT and pSR aCAT carry the CAT gene under the control of the SV40 promoter. pCHL4 and pWEN-CAT carry the CAT gene which is controlled by the promoter from human T-cell leukemia virus type I (HTLV-1) and polyomavirus, respectively. Cell extracts were prepared 48 hr after electroporation, and incubated with ['4C]chloramphenicol (60 mCi/mmol; Du Pont NEN) and acetyl coenzyme A (Pharmacia). The chloramphenicol and its acetylated forms were extracted with cold ethyl acetate, separated on silica gel thin-layer plates (Merck). Their radioactivities were counted with Radioisotope Scanning System (AMBIS, San Diego), and also autoradiographed on X-ray film (Kodak). RESULTS

EBNA-2 Expressionin BJAB and DG75 Cells Cell surface antigens of several EBV-negative human B-lymphoma cell lines were examined by flowcytometry analyses (Table 1). BJAB and DG75 cells were chosen as recipient cells for the analyses of an effect(s) of EBNA-2 on B cells in this study, because expression of the cell surface markers CD21 (B2), CD23 (H 107) and CD25 (anti-IL2R) was undetectable on their surface.

CD25 INDUCTION

IN B CELLS

EXPRESSING

EBNA-2

483

Table 1. Surface markers on four EBV-negative B cell lines and LCL-C4 which was established by infection with EBV (B95-8)

A

pKCRE2

B

pSVLMPneo

C

pC43

Fig. 1. Schematic representation of construction of the recombinant plasmids. A, pKCRE2 for EBNA-2 expression; B, pSVLMPneo for LMP expression; C, pC43 for EBNA-1 expres-

sion. The inserted DNA fragments containing EBNA-2, LMP, EBNA-1 and the neomycin phosphotransferase gene (neo) are indicated in the open bars attached to the plasmid circles. Arrows inside the bars indicate the orientation of transcription of the genes. The vector plasmids are denoted inside the plasmid circle. The shaded arcs and bars represent the SV40 sequences. The hatched arcs represent the Eco-gpt sequences, the small open circles indicate the replication origin of pBR322. ApR stands for the ampicillinase gene.

The EBNA-2-expressing plasmid pKCRE2 was constructed as depicted in Fig. 1A. The plasmid pC43 carrying EBNA-1 was constructed as shown in Fig. 1C. To test whether these two plasmids confer EBNA-2 and EBNA-1 expression in B cells, respectively, DG75 cells were transfected with pKCRE2 or pC43, and analyzed by ACIF 48 hr post-transfection. The transient expression of EBNA-2 (Fig. 2B) was

484

S. HARADA

AND K. YANAGI

as intense as that of EBNA-1 (Fig. 2A) in the ACIF analysis with the human EBNA-positive serum KM. To produce cell clones which constitutively express EBNA-2, the co-transfection of pKCRE2 with pSV2gpt at the molar ratio of 10 to 1 into DG75 and BJAB cells was carried out. After selective culture of the co-transfected cells for two to four weeks post-transfection in the presence of mycophenolic acid (MPA), 15% of MPAresistant cell clones were confirmed to express EBNA-2 by the ACIF analyses with the KM serum. Vector-transfected-control cell clones did not react with this serum (Fig. 2E). The EBNA-2 of the expressing cell clone was distinctively detected in the nuclei as shown in Fig. 2D, but its intensity was not as strong as that of the transiently expressed EBNA-2 shown in Fig. 2B. The immunofluorescence in the cell clone was punctate with a range of intensity, and was not different in the pattern between the BJAB- and DG75-derived cell clones. This pattern of EBNA-2 immunofluorescence differed from that of EBNA-1 fluorescence which was dispersed throughout the nuclei as presented in Fig. 2C. These patterns of EBNA-2 fluorescence were in agreement with those of the EBNA-2 fluorescence which had been reported by other laboratories (11, 30). These cell clones were further examined by the immunoblot analyses with the serum KM. A predicted 84 kDa polypeptide was detected in the cell lysates of EBNA-2-expressing cell clones (Fig. 3). A control EBNA-2 band of the same size in a lymphoblastoid cell line LCL-C4 is presented in the lane 7 in Fig. 4 together with EBNA-1 and the other larger EBNA protein bands. We examined the growth rate of the constitutively EBNA-2-expressing cell clones to see whether they had any growth advantage at low concentrations of fetal calf serum in the culture medium, but little difference in the cell growth rate or cell saturation density between them and the parent cells was observed (data not shown). Next, we attempted to examine whether EBNA-2 has a trans-activating capability on several viral promoter-regulatory sequences. The plasmids pSV2CAT, pCHL4 and pWEN-CAT were used for the expression of an indicator CAT gene under the control of the promoter-regulatory sequences from SV40, HTLV-1 and polyomavirus, respectively. No up-regulation of the activity of the promoter of HTLV-1 or polyoma was indicated. The pSV2CAT activity in the constitutively EBNA-2expressing cell clone D50 was 9:4%, whereas it was 2.8% in the vector-transfected control cell clone E3 (Fig. 4B). On the other hand, in the transiently EBNA-2expressing BJAB cells which were transfected with the EBNA-2-expressing pKCRE2, both pSV2CAT and pSR aCAT, in which the CAT gene was controlled by the SV40 early promoter, were scarcely enhanced (Fig. 4A). This little or no effect in the transiently EBNA-2-expressing cells in this study is well consistent with the experimental result of EBNA-2 on the activity of SV40 promoter in pSV2CAT mentioned by Wang et al (49). Cell Surface Antigenson EBNA-2-Expressing Cells We searched the EBNA-2-expressing cell clones derived from BJAB and DG75 for a possible alteration(s) in the expression of a cell surface marker(s) by flowcyto-

CD25 INDUCTION

IN B CELLS

EXPRESSING

EBNA-2

485

Fig. 2. Anti-complement immunofluorescence (ACIF) analyses. (A and B): DG75 cells which were transfected with pC43 for EBNA-1 expression (A) or pKCRE2 for EBNA-2 expression (B). The cells were fixed at 48 hr post-electroporation. (C-E): ACIF staining of the constitutively EBNA-expressing cell clones derived from DG75. The EBNA-1-expressing cell clone DGA6B (C), the EBNA-2-expressing cell clone DGB47 (D), and the control cell clone E3 (E), which were established by transfection with pC43, pKCRE2, and the vector pKCRH2, respectively. The human EBNA-positive serum, KM, was used as antiserum against EBNA-1 and EBNA-2 for the analyses. All the microscopic photographs were taken by 400-fold magnification.

metry analyses. The cell clones were analyzed for expression of the eight cell surface markers, CD10, CD19, CD20, CD21, CD23, CD25, HLA-DR, and HLA-DQ (Table 2). Monoclonal antibodies which were used to detect these surface markers are displayed in Table 2. The cell clones E3 and Y20 which had been made by transfection with the vector pKCRH2 were analyzed as negative control cells. There was no significant difference in the seven cell surface markers, CD20, CD 19, CD21, CD10 (CALLA), CD23, HLA-DR and HLA-DQ, between all the examined EBNA-2-expressing cell clones and the control cell clones (Table 2). It is noteworthy that CD21 and CD23 were undetectable on all the examined cell clones as in the parent cells (Table 1). The LCL-C4 cell line, which was established by

486

S. HARADA

AND K. YANAGI

Fig. 3. Immunoblot analyses. Lane 1 (Raji) and lane 7 (LCL-C4), EBV-genome positive cells; lanes 2-6, the BJAB-derived cell clones; lanes 8-10, the DG75-derived cell clones. Lane 6 and lane 10 are control cell clones which were made by transfection with the vector DNAs only; lanes 2-5, 8 and 9 are the ACIF-positive EBNA-2-expressing cell clones established by the co-transfection of pKCRE2 and pSV2gpt (Fig. 1). Proteins were separated on 10% SDS-polyacrylamide gel (lanes 1 to 6) and on 7.5% SDS-polyacrylamide gel (lanes 7 to 10). The human EBNA-positive serum, KM, was used for the analyses. Arrows indicate EBNA-2 bands.

B95-8 infection of B lymphocytes from cord blood, was expectedly CD23-positive as indicated by the reaction with the anti-CD23 monoclonal antibody H107 (Table 1). In order to examine whether up-regulation of CD23 mRNA was induced in the EBNA-2-expressing cells, northern blot analysis was performed, but no increase in the level of CD23 mRNA in two BJAB-derived cell clones, D50 and D75, was observed (data not shown). It was indicated, however, by the flowcytometry profiles in Fig. 5 that CD25 was expressed in DGB43, one of the examined cell clones derived from DG75, in contrast to the control cell clone Y20. Next, we examined whether CD23 and CD21 could be induced in DG75 cells by co-expression of EBNA-2 and LMP. The LMP-expressing plasmid pSVLMPneo was transfected in combination with pKCRE2 or alone into DG75 cells, then the transfected cells were selected by growth in the presence of G418. Through screening by ACIF of the G418-resistant cell clones, we obtained EBNA-2-expressing cell clones. Expression of LMP on the cell clones was analyzed by the flowcytometry analysis with the anti-LMP monoclonal antibody S12 in which the cells were prefixed with formaldehyde according to the method of Mann et al (21). Then, expression of the eight cell surface antigens on the cells which expressed both EBNA-2 and LMP or LMP alone was quantitatively analyzed (Table 2). An increased

CD25

INDUCTION

IN

B CELLS

EXPRESSING

EBNA-2

487

A

B

Fig. 4. CAT assay of viral promoters activity in BJAB cells which transiently expressed EBNA-2 by co-transfection with EBNA-2 and CAT genes (A) and in the constitutively EBNA-2-expressing cell clone 1)50 (B). (A): BJAB cells were co-transtectect ny eiectroporation with the EBNA-2 plasmid pKCRE2 (E2) or the vector plasmid pKCRH2 (KCR), in combination with CAT gene which is regulated by the SV40 promoter (pSV2CAT or pSRa. CAT) or not regulated by the SV40 enhancer (pA 10CAT). BJAB cells were cotransfected in a similar fashion in combination with CAT gene which is controlled by the HTLV-1 promoter (pCHI.4) or the polyomavirus promoter (pWEN-CAT). (B): The EBNA-2-expressing cell clone 1)50 and the control cell clone E3 were transfected with pA 10CAT, pSV2CAT, pCHI.4 or pWEN-CAT. The percent acetylation of pSV2CAT was 9.4% and 2.8(% in D50 and E3 cells, respectively.

S. HARADA

488

Table 2.

AND K. YANAGI

Expression of surface markers on BJAB- and DG75-derived cell clones which expressed EBNA-2 and/or LMP

1, except for the reaction of MAb S12 with cells which were fixed by formaldehyde. The figures indicate percentages of surface antigen-positive cells.

expression of CD21 and CD23 such as reported from other laboratories (8, 48) was hardly observed on any cell clone which we made and examined in this study. It should, however, be noted that the experimental data does not necessarily exclude a possibility of CD21 increase in the cell clones, since the epitope to the anti-CD21 monoclonal antibody B2 was not detected on several CD21-positive B cell lines (3). It was found by the flowcytometry that A102 cells, which expressed both EBNA2 and LMP, were positive for CD25 (Fig. 5 and Table 2). Forty percent of the A102 cells were stained with the monoclonal antibody S12, and it was higher than the percentages of S12-stained cells in the other LMP-positive cell clones (Table2). CALLA (CD10) was not detected on A102 but was retained on DG75 and the

CD25

INDUCTION

IN

CD25

EBNA-2

489

Y20

C18

A102

CD10

5.

EXPRESSING

DGB43

CD25

Fig.

B CELLS

C18

A102

Flowcytometry

expressing

cell

clones

analyses derived

of surface from

DG75.

markers DGB43

CD25, expressed

CD10

and

EBNA-2

LMP

of the

EBNA-2-

solely,

A102

expressed

dually EBNA-2 and LMP. Y20 and C18 are the control cell clones which were made by transfection with vector DNAs. Monoclonal antibodies (MAbs) anti-IL-2R, and anti-CALLA were used to stain and quantify CD25, CD 10, respectively. The living cells were treated with anti-IL-2R and anti-CALLA antibodies. The solid lines represent the fluorogram of the cell clones with the number of cells on Y-axis and fluorescence intensity by log scale on X-axis. The dotted lines represent fluorescence of the cells which were treated with FITCconjugated mouse IgG1 as negative control.

control C18 cell (Fig. 5 and Table 2), indicating that CALLA was totally suppressed in the LMP-expressing A102 cells as in LCL-C4 (Table 1). DISCUSSION

BJAB- and DG75-derived cell clones which constitutively expressed EBNA-2 were made and analyzed in this study (Figs. 2 and 3, Table 2). The two B cell lines, BJAB and DG75, scarcely expressed the three cell surface markers CD21 (B2), CD23 (H 107), and CD25 (anti-IL-2R) (Table 1), and were useful for the examination of possible induction of the surface markers. The up-regulation of CD23 by EBNA-2 was hardly detected in the BJAB and

490

S. HARADA

AND K.

YANAGI

DG75 cell clones in this study (Table 2) . CD23, referred to also as Blast 2 or EBVCS, is one of the B cell activation antigens, and is known to appear on the cell surface of EBV-infected B lymphocytes (41). Aman et al reported that an increase in CD23 was detected in U698 cells but not in BJAB cells in which EBNA-2 gene was transfected (3). Their observation on the BJAB cells is in agreement with our result of no increase in CD23 on the EBNA-2-transfected BJAB cells. On the other hand, it was reported that EBNA-2 increased CD23 expression in Louckes cells (47) and in a BL cell line containing the EBV P3HR-1 genome (8). The apparent discrepancy between these reports in the up-regulation of CD23 by EBNA-2 is thought to be due to a possible difference in the response to EBNA-2 between the EBV-negative B cell lines used as recipient cells. It is also a possibility that the level of stable EBNA-2 expression in the cell clones was critical for the induction of CD23 up-regulation, and the critical level varied from one cell line to another between the EBV-negative B cell lines used. CD25 is the interleukin-2 receptor (IL-2R) of molecular weight 55,000 and has been referred to IL-2R (Tac) or IL-2R/p55 (Tac). It should be stressed that an induction of CD25 was found for the first time in this study by the flowcytometry analyses of the EBNA-2-expressing cell clone DGB43 and the EBNA-2- and LMPco-expressing cell clone A102 (Table 2, Fig. 5). These two cell clones were both derived from the DG75 cell line. The intensity of CD25 flowcytometry and the percentage of CD25-positive cells in the two cell clones, DGB43 and A102, were not high (Fig. 5). Relevantly, Waldmann et al reported that the level of IL-2R expression on activated B cells was 5- to 10-fold less, even several hundreds-fold less in some cases, than that on activated T cells (46). Hence, the depicted little reactivity with the anti-CD25 monoclonal antibody of major populations of cells of the two cell clones (Fig. 5) may be due to their insufficient abundance of the cell surface antigen to be detected by the flowcytometry analysis. In any case, the results in this report indicate that EBNA-2 potentiates an up-regulation of CD25 in at least about onethird of the transfected B cells. Relevant to this result, Waldmann et al reported that a proportion of Tac-positive cells of one cell clone, which they cloned by limiting dilution from Tac-positive B cell lines, was approximately 5%, and the proportion increased to about 30% or higher upon an induction of the surface antigen with interleukin-2 (IL-2) (46). A question of why only the two cell clones and not all of the EBNA-2 cell clones, and about 30% but not 100% of them, showed the induction of CD25 may be raised. A possible explanation is that a limited number of cell clones expressed EBNA-2 over a certain threshold level which is required for the CD25 induction. As a matter of fact, the ACIF intensity of EBNA-2 in DGB43 (data not shown) was as strong as that of the transient EBNA-2 expression, which is shown in Fig. 2B, being considerably higher than those of the other EBNA-2expressing cell clones shwon in Fig. 2D. Another possibility is that a pathway(s) is so complicated that EBNA-2 expression does not necessarily result in the induction of CD25 in all EBNA-2 positive cell clones, depending on varied physiological conditions of recipient cells at and around the moment of transfection. In addition, it was implied that a combined effect of EBNA-2 and LMP contributed to the CD25

CD25

INDUCTION

IN B CELLS

EXPRESSING

491

EBNA-2

enhancement in A102. Relevant to the possibility, it was reported that CD23 was enhanced in the Loucks cell line by a cooperative effect of EBNA-2 and LMP (48), although CD23 was not detected in the A102 cells (Table 2). It is noteworthy that the CD25 induction on these two cell clones took place without an up-regulation of CD23, suggesting that a physiological pathway for the induction of CD25 by EBNA-2 proceeds independently of the CD23 enhancement. The disappearance of CALLA concomitant with LMP expression which was observed on the A102 cells is in agreement with the report by Wang et al (48). It has been recently reported that EBNA-2 exerted a simulatory effect on the level of c-fgr RNA (19) and an interference with the interferon-induced anti-proliferative response (2). It has been also reported that EBNA-2 transactivated the expression of LMP1 and the terminal protein (LMP2) which are encoded by the EBV genome (1, 14, 49, 50). Thus, EBNA-2 is considered to hold multiple effects on the expression of a variety of cellular and viral genes. This study has added another effect, i.e., an up-regulation of CD25 in B cells, to the list of the effects on cells induced by EBN-2. It is of particular interest that CD25 was detected in some activated B lymphocytes including EBVA-transformed B cells (46). CD25, which has been referred to IL-2R/p55 (Tac), is known to be expressed at a high level on plasma membrane of T cells from adult T-cell leukemia (ATL) patients and play an important role in T cell proliferation (37). The autocrine system of IL-2 and IL2R is considered to play a major role in the development of ATL by HTLV-1. In addition, it has been recently reported that an EBV-infected B cell subclone had a large amount of IL-2R and secreted an autocrine factor (44, 45). The autocrine factor secreted by the B cell subclone has been recently indicated to be identical to a T cell-derived factor, ADF, which was found to induce IL-2R/p55 (Tac) or CD25 on T cells (39, 45). Hence, based on the multi-potential of EBNA-2 discussed above, we present a hypothesis that EBNA-2 is able to induce the cellular autocrine factor which is involved in the CD25 up-regulation. We clones,

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Foundation.

REFERENCES

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Induced CD25 expression in a human B-lymphoma cell line transfected with the Epstein-Barr virus nuclear antigen 2 gene.

Two EBV-negative human B-lymphoma cell lines, BJAB and DG75, were transfected with an Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) gene, which ...
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