Virus Research 11 (1988) 209-225 Elsevier

209

vm 00445

Differences in Epstein-Barr virus (EBV) receptors expression on various human lymphoid targets and their significance to EBV-cell interaction

Rino Stocco, Guy Sauvagea~ and Jo& Menezes Luboratoy of Imrn~~~roio~, department of Microbiolo~ and Immunolo~ and Pediatric Research

Center, U~~ue~i~ of Montreai and SteJustine Hospital, Montrprri, Qukbec, Cam&

(Accepted 8 June 1988)

This study was aimed at quantitating, by means of fluorescence-activated cell sorter (FACS), EBV binding to different types of target cells, and at learning about a possible relation between EBV receptor density and the fate of cell-surface bound virus. We used fluoresceinated virus preparations of two strains of EBV (B95-8: lymphocyte tr~sfor~ng strain; P3HR-1: non-tr~sfo~ng strain) to analyze quantitatively the expression and density of EBV receptors on different human lymphoid cell lines and on B lymphocytes from both EBV-seropositive and -seronegative donors. FACS analysis was also used as a tool to approximate the cell surface area of the different lymphoid cells exam@d. Our results indicate that: (a) after accounting for the difference in cell surface dimensions, the fluorescence intensity of EBV-bound Raji (a B line) cells was three to four tunes higher per unit area than that of EBV-bound fresh B lymphocytes from an EBV-seropositive donor; (b) Molt-4 (a T line) cells bound about 21-fold less P3HR-1 EBV and 6-fold less B95-8 EBV than Raji cells per unit area; (c) B lymphocytes from EBV-seronegative adult donors bound only about one third as much virus as B cells from seropositive in~~du~s; (d) two B lymphocyte sub-populations can be identified in the periph- eral blood in regard to their ability to bind EBV, regardless of the EBV antibody status of the donor; (e) the EBV receptor on Molt-4 cells appears st~ctur~ly different from the one found on Raji cells since EBV binding to Molt-4 cells was not blocked by a monoclonal antibody (OKB7) specific to the complement receptor

Correspondence to: Dr. Jo& Menezes, Laboratory of Immunovirology, Sk-Justine Hospital, 3175, Cote Sainte-Catherine, Mont&al, Qudbec, Canada, H3T 1CS.

0168-1702/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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(CR2). Further, in contrast to Raji cells, Molt-4 expressed a differential binding activity for each of the two EBV strains used. Taken together, the important differences observed in regard to EBV attachment to various targets also appear to relate to the fate of cell-surface bound virus: i.e., virus penetration might be determined, at least in part, by the density of EBV receptors on the target cell surface; thus the receptor density may play a major role in viral infection.

Epstein-Barr virus; Receptor expression; Human lymphoid cell

Introduction

Epstein-Barr virus (EBV) is a human lymphotropic herpesvirus that causes infectious mononucleosis (Henle et al., 1968; Niederman et al., 1968) and its genome is regularly associated with two human cancers: Burkitt’s lymphoma (de The et al., 1978; zur Hausen’ and Schulte-Holthausen, 1970) and undifferentiated nasopha~ngeal carcinoma (Glaser et al., 1976; zur Hausen et al., 1970). The host range of EBV is determined by specific interactions between the viral envelope and EBV receptors (EBV-Rs) present on target cell membranes. EBV-Rs have been detected mainly on human B lymphocytes, especially those bearing surface IgM, and on established lymphoid cell lines (LCLs) of B cell lineage (Jondal and Klein, 1973; Katsuki et al., 1977; Menezes et al., 1976). Certain LCLs of non-B origin such as the T cell line, Molt-4, also express EBV-Rs (Menezes et al., 1977; Pate1 and Menezes, 1981). Interestingly, EBV binding to Molt-4 cells is followed neither by virus penetration as shown by electron microscopic observations, nor by expression of any of the known markers of EBV infection detectable by immunofluorescence (Menezes et al., 1977; Pate1 and Menezes, 1981; Shapiro et al., 1982). A similar situation was also found with other LCLs which bear EBV-Rs but are not infectable by the virus (Pate1 and Menezes, 1981). Two experimental model systems developed in our laboratory showed that concanavalin A-mediated binding of EBV to EBV-R negative cells was not followed by virus infection (Khelifa and Menezes, 1982a), whereas Sendai virus envelope-mediated EBV binding resulted in infection of resistant cells (Khelifa and Menezes, 1983). The different outcome of EBV binding in these two model systems was ascribed to the presence of the Sendai virus fusion factor Frl and led us to consider that EBV envelope-cell membrane fusion in susceptible cells may be controlled by an as yet unknown cellular mechanism (Khelifa and Menezes, 1982a). This conclusion is further supported by the observa- tions of Shapiro et al. (1982) showing that Molt-4 cells become susceptible to EBV infection after implantation on their plasma membrane of EBV-Rs containing fragments of Raji cell membranes. Further, in contrast to the known mode of EBV penetration by envelope fusion in LCLs (Menezes et al., 1977; Seigneurin et al., 1977), Nemerow and Cooper (1984) reported that this virus enters normal B lymphocytes by endocytosis and subsequent reenvelopment in endocytic vesicles. It seems therefore that EBV envelope fusion with the cell membrane of susceptible B

211

LCLs is determined, at least partly, by some cellular factor(s) present on these LCLs but not on normal B lymphocytes or on resistant LCLs. These observations suggest that there are different mechanisms available for EBV entry in various target cells and that it is at the level of the target cell membrane that the fate of cell surface-bound EBV is determined (i.e. mode of penetration or lack of penetration of cell-bound EBV).

In 1976, Jondal and Klein showed that the EBV-R could be detected on the surface of cell lines also bearing the receptor for the C3 fraction of complement (Jondal et al., 1976). This observation was further supported by the findings that EBV and C3 receptors were co-expressed on the surface of B cells (Jondal et al,, 1976), that they co-capped (Yefenof et al., 1976) and that stripping of the C3 receptor from the cell surface abolished subsequent binding of EBV (Yefenof and Klein, 1977). More recently, using ceils of a B (Raji) LCL, it was shown that this relation is due to the fact that the receptor for C3d (or CR.2) represents also the receptor for EBV (Fingeroth et al,, 1984; Frade et al., 1985; Nemerow et al., 1985). It is, however, unclear whether the EBV-R found on non-B, specially on T LCLs has any such direct relation to CR2.

We undertook the present study with three main objectives in mind: (a) to quantitate EBV binding to different types of target cells, (b) to learn whether there is any relation between EBV-R density and the fate of cell-surface-bound virus, and (c) to analyse further the difference between the EBV-R on Molt-4 cells and on other LCLs. We used the fluorescence-activated cell sorter (FAGS) to quantitate the binding of fluorescein-labelled EBV (FITC-EBV) to various target cell types pre- senting the different outcomes of EBV binding referred to above. We examined: (a) quantitative differences in virus attachment among various types of target lymphoid cells using both the lymphocyte transforming (B95-8) and non-transforming (P3HR- I) strains of EBV; (b) the relation between cell surface area and the amount of virus bound to target cells using LCLs and peripheral blood B lymph~yt~s; and (c) the unique ~tera~tion between the two EBV strains and their cellular receptor on Molt-4 cells. Our data illustrate the existence of important qualitative and quantita- tive differences in EBV-R expression on the various cell lines used. These dif- ferences appear pertinent in regard to the various types of early EBV-target cell interactions known. Furthermore, the data also suggest that the differential binding of EBV strains and lack of binding of anti-CR2 monoclonal antibody to Molt-4 cells result from the fact that the cellular receptor site involved in EBV binding to these T cells is of a different nature than the one from Raji cells.

Materials and Methods

The LCLs used in these studies were cultured at 37’ C in RPMI-1640 medium (RPMI) supplemented with 10% heat-inactivated fetal bovine serum and antibiotics as described previously (Pate1 and Menezes, 1981). The cultures were incubated in

212

the presence of 5% CO,, fed twice weekly and readjusted to 2 x lo5 cells/ml at each feeding.

Lymphocyte purification

Peripheral blood lymphocytes (PBL) from healthy adults were prepared by centrifugation on Ficoll-hypaque density gradient. B lymphocytes were separated as follows: PBL were permitted to settle in tissue culture flasks for 1 h to remove monocytes/macrophages: nonadherent cells were removed and treated with an appropriate dilution of OKT3 monoclonal antibody (Ortho Pharmaceuticals, Rari- tan, NJ) for 20 min at 37’ C. The 0KT3 labelled T lymphocytes were then lysed by adding 50 ~1 of pooled rabbit complement and then incubating the cells for 1 h in the same conditions as above. The rabbit complement had no cytotoxic effect when incubated alone with lymphocytes.

Characterization of B lymphocytes with the 0KB7 monoclonal antibody

Ortho 0KB7 murine monoclonal antibody, which labels over 95% of peripheral blood B lymphocytes, was used to determine the degree of purification of our B lymphocyte preparations. One million B lymphocytes mixed with 50 ~1 of ap- propriately diluted OKB7 monoclonal antibody were incubated for 20 mm at 4” C with frequent agitations. Cells were then labelled with 50 ~1 of fluoresceinated goat anti-mouse IgG, F(ab’), fraction (Cappel Laboratories, West Chester, PA) and washed three times with RPMI. The cell pellet was then resuspended in a 1% solution of paraformaldehyde in phosphate-buffered saline (PBS, pH 7.2). B lymphocytes were also characterized by the presence of surface immunoglobulins using fluoresceinated polyvalent antibodies directed against human IgG, IgM and &A (from Cappel Laboratories).

Virus preparation

EBV-producing cell lines P3HR-1 and B95-8 were used as a source of non-trans- forming P3HR-1 (PEBV) and lymphocyte-transforming B95-8 (BEBV) strains of EBV, respectively, as described previously (Menezes et al., 1975; Pate1 and Menezes, 1981). Briefly, supernatants of these cultures were clarified by low-speed centrifuga- tion and membrane filtration (0.45 pm Nalgene units; Nalgene Labware Depart- ment, Rochester, NY). Virus was then pelleted at 45 000 x g for 90 min and gently resuspended in cold PBS to obtain the desired concentration (> 400 times). Virus titers were determined as described (Menezes et al., 1975; Pate1 and Menezes, 1981).

Virus jluoresceination

Concentrated EBV was labelled with fluorescein isothyocyanate (FITC, Isomer I, Sigma Chemical Co., St. Louis, MO), as previously described (Khelifa and Menezes, 1982b). Briefly, 1 volume of a 2 mg/ml solution of FITC in 0.5 M carbonate-bi-

213

carbonate buffer (pH 9.5) was added, while shaking, to 9 volumes of concentreated PEBV or BEBV. The mixture was kept in the dark at room temperature for 1 h, with frequent agitations. The labelled virus (FITC-EBV) was separated from unbound FITC, cellular debris and proteins, using a Sephadex G-25 column, as described (Khelifa and Menezes, 1982b).

Virus binding assay

Two FITC-EBV preparations (one for each EBV strain) of comparable titer and capable of saturating EBV receptors, as determined in preliminary binding assays using Raji cells (Khelifa and Menezes, 1982b), were used for these studies. Fifty ~1 of FITC-EBV (PEBV or BEBV) and 50 ~1 of PBS were added to a pellet of 5 X lo5 cells. The mixture was incubated for 45 min in the dark at 4O C with frequent agitation. The choice of this incubation period was based on previous observations showing that maximal EBV attachment does occur prior to 45 min of incubation in all the cell lines tested (Khelifa and Menezes, 1982b; Pate1 and Menezes, 1981). The cells were then washed thrice with cold PBS and the cell pellet was resuspended in approximately 0.7 ml of a 1% paraformaldehyde solution in PBS and analyzed by FACS. The specificity of EBV binding was determined in a blocking assay in which FITC-EBV was preincubated with an anti-MA (membrane antigen) monoclonal antibody (72Al) specific to EBV gp250 which is known to neutralize EBV (Hoffman et al., 1980) and to inhibit EBV binding (Stocco and Menezes, submitted for publication).

Competitive binding assay

This assay was performed on both Molt-4 and Raji cells by incubating a pellet of 5 x lo5 cells with 50 ~1 of either one of the two strains of EBV (PEBV or BEBV) for 1 h at 4O C. After three washes with PBS, 50 ~1 of FITC-labelled BEBV or FITC-labelled PEBV were added to the cells and the preparation was then in- cubated for 1 h at 4 o C. After this incubation period and subsequent three washes with PBS, the cell pellets were resuspended in 0.5 ml of PBS-paraformaldehyde 1%.

CR2 assay

The 0KB7 monoclonal antibody was also used in order to assess the presence of C3d receptors (CR2) on the surface of target cells tested. Ten ~1 of undiluted 0KB7 monoclonal antibody were added to 1 X lo6 cells in 400 ~1 of PBS and incubated for 45 min at 4°C. Then, 50 ~1 of an appropriate dilution of F(ab)‘, sheep anti-mouse IgG-FITC (Bio/Can, Toronto) preparation was added to the cells and incubated for 45 min in ice. The cell pellet was then resuspended in 0.5 ml of PBS-paraformalde- hyde 1%. Controls included cells incubated with an unrelated monoclonal antibody (before adding the labelled anti-mouse antibody) and cells incubated with the FITC-conjugate alone.

214

EB V blocking assay

One million Raji or Molt-4 cells were incubated with 25 ~1 of OKB7 monoclonal antibody in 1 ml of PBS for 45 min at 4°C. Following three washes with PBS, 100 ~1 of FITC-EBV were added to the cell pellet and incubated for 45 min in ice in the dark. After three more washes with PBS the cell pellet was then resuspended in 0.5 ml of PBS-paraform~dehyde 1%. The percentage of inhibition of EBV binding by 0KB7 was calculated by the following formula:

% pos. cells with FITC-EBV - % pos. cells with 0KB7 and FITC-EBV x loo % pos. cells with FITC-EBV

Use of FA CS

Calibration procedures and cell analysis were performed using a FACS-III (Becton-Dickinson FACS system, Sunnyvale, CA) connected to a realtime acquisi- tion display system (Stewart and Price, 1986). The ion laser beam (Model 164-05, Spectra Physics, ~ount~n View, CA) was tuned for m~um output at 488 nm, and typically set for 200 nW light output. For detecting fluorescence of micro- spheres or fluoresceinated cells, a 520-560 nm filter package was placed between the beam-splitter and the ‘green’ PMT (FACS III: Fl-1 parameter). ‘Fluoresbrite’ carboxylated microspheres (0.84 & 0.023 pm diameter, lot no. 24513, catalogue no. 15702, Polysciences Inc., Warrington, PA) were used for calibrating the log, fluorescence settings. According to our standardization procedure, one unit of fluorescence corresponded approximately to 3 x lo4 fluorescein molecules. To calibrate the small angle light scatter, beads of 4.28 pm of diameter (surface = 123.9 pm2) were analyzed at channel 26 with a gain of 16.

We used 3-parameter, realtime acquisition and display analysis and storage of data obtained from flow cytometric (FACS III) analysis of cells. Microcomputer hardware and software were utilised throughout the design. Software routines for the operator’s use were written using a l&bit version of the FORTH language (68K-FORTH, Empirical Research Group Inc., Milton, VA). The realtime routines were written in assembly- or machine-language for the MC6800, a 16/32 bit microprocessor (Motorola Semiconductors Inc., Austin, TX).

Results

FACS analysis of EBV receptor expression human lymphoid cell lines

Raji cells were used as receptor-positive controls (Jondal et al., 1976; Khelifa and Menezes, 1982b; Menezes et al., 1977; Pate1 and Menezes, 1981). Optimal con- centrations of FITC-EBV were determined by both fluorescence and electron microscopy (Khelifa and Menezes, 1982a, b; Pate1 and Menezes, 1981) and con- firmed by preliminary tests using FACS. As it was previously shown that both

215

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Fig. 1. FACS profile of EBV binding to Raji cells. Top: background fluorescence was determined with Raji cells sham-treated with PBS; middle: Raji cells incubated with FITC-PEBV; bottom: Raji cells

incubated with FITC-BEBV.

BEBV and PEBV bind equally to Raji cells (Khelifa and Menezes, 1982b; Pate1 and Menezes, 1981), FITC-EBV concentrations of both virus strains giving optimal and identical profiles of EBV binding to these targets were used throughout this study (Fig. 1).

The 1301 cell line which does not bear EBV-Rs on its surface (Khelifa and Menezes, 1982b; Menezes et al., 1977; Pate1 and Menezes, 1981) did not show any significant increase above the background level (i.e. cells without FITC-EBV) in its fluorescence intensity following incubation with fluoresceinated virus (Fig. 2). These results of FITC-EBV binding tests using Raji and 1301 cells indeed confirm that the fluoresceination procedure does not alter the cell binding specificity of EBV, ruling out fluorescein-induced non-specific binding of virus to cells. Furthermore, the fluoresceination of EBV did not alter its strain-specific differential binding to the EBV receptor-positive T cell line Molt-4 (Fig. 3), thus confirming the results

216

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l.

t

N

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B

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FLUWESCENCE INTENSITY

Fig. 2. FACS profile of EBV binding to 1301 cells. Top: background fluorescence was determined with 1301 cells sham-treated with PBS; middle: 1301 cells incubated with FITC-PEBV; bottom: 1301 cells

incubated with FITC-BEBV.

reported earlier using a different approach (i.e. i~unofluorescence; Pate1 and Menezes, 1981). Molt-4 cells bound approximately two times more FITC-BEBV than FITC-PEBV since the fluorescence intensity obtained with the former was two times higher than that observed with the latter (Fig. 3). This result was not due to a difference,in the extent of fluoresceination or in the virus concentration in the two EBV preparations used since, as shown above, under the same experimental conditions, both preparations gave the same FACS profile (i.e. identical fluores- cence intensity) with Raji cells. To demonstrate the specificity of FITC-EBV binding to these targets, we used the 72Al anti-MA monoclonal antibody which has been found, in a previous study, to inhibit EBV binding to EBV-R positive cells (Stocco and Menezes, submitted for publication),

217

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Fig. 3. FACS profile of EBV binding to Molt-4 cells. Top: background fluorescence was determined with Molt-4 cells sham-treated with PBS; middle: Molt-4 cells incubated with FITC-PEBV; bottom: Molt-4

cells incubated with FITC-BEBV.

Comparison of EBV receptor density on Raji cells, Molt-4 cells and fresh peripheral blood B lymphocytes from EBV-seropositive and -seronegative donors

FACS analysis allows us to evaluate another parameter in the binding of viruses to target cells: the cell surface. In an attempt to learn more about the cell surface characteristics that might contribute to cellular mechanisms controlling the fate of cell-bound EBV, we compared the relative densities of EBV-Rs on three types of target cells. These targets were chosen to represent the three known possible outcomes of EBV binding: (a) penetration by viral envelope-cell membrane fusion in B lymphoblastoid (e.g. Raji) cells (Menezes et al., 1977; Seigneurin et al., 1977); (b) penetration by receptor-mediated endocytosis in peripheral blood B lymphocytes

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TABLE 1

COMPARISON OF FITC-EBV BINDING TO RAJI, MOLT-4 AND PERIPHERAL BLOOD B

LYMPHOCYTES

Cell Virus Small angle CSA MFI b MFI/CSA ratio ’

type strain light scatter (pm’) a

Raji PEBV 55 524.2 725 1.383 Raji BEBV 50 416.5 550 1.154 B cell (s+) d PEBV 35 333.6 110 0.330 B cell (sf ) BEBV 35 333.6 125 0.375 Bcell(s-)d PEBV 35 333.6 35 0.105 B cell (s-) BEBV 35 333.6 45 0.135 Molt-4 PEBV 65 619.5 40 0.065 Molt-4 BEBV 65 619.5 115 0.186

a CSA: cell surface area.

b MFI: For Raji and Molt-4 cells, the figures given here represent modal fluorescence intensity in

arbitrary units; for B cells, because of their bimodal distribution (see also Fig. 4), the numbers

represent the mean fluorescence intensity in arbitrary units.

’ MFI/CSA ratio: This ratio represents the relative EBV-R density on the cell surface.

d s+: B lymphocytes from two EBV-seropositive adult donors; s-: B lymphocytes from two EBV-

seronegative adult donors. These B cell preparations contained z 90% of B cells (as determined by

their reactivity with OKB7 monoclonal antibody and with anti-Ig antibodies).

(Nemerow and Cooper, 1984) and (c) lack of penetration in Molt-4 cells (Menezes et al., 1977). Raji cells, Molt-4 cells, or peripheral blood B lymphocytes from healthy EBV-seropositive and -seronegative donors were treated with FITC-EBV of the indicated strain and subjected to FACS analysis. Using the values of small-angle light scatter given in Table 1, we calculated the mean surface area of these cells, according to the calibration settings indicated in Materials and Methods. The ratio of the MFI of the cells and their mean surface area was taken as a relative estimation of the mean EBV-R density. As calculated from the results shown in Table 1, the mean receptor density was approximately 4.2 and 3.1 times higher on Raji cells than on peripheral B lymphocytes of seropositive donors for FITC-PEBV and FITC-BEBV, respectively. Moreover, when FITC-EBV binding to B cells was analyzed in relation to the donor’s EBV antibody status, it was found that B lymphocytes from the two seropositive individuals had an EBV-R density about three times higher than that of B cells from the two seronegative donors, and this for both strains of EBV (Table 1); the size of B lymphocytes from both groups was identical, however. These results, although obtained with a limited number of donors, nevertheless suggest that B cells from adult seropositive persons possess a higher EBV-binding capacity than those from the seronegative adult donors. It may also be pertinent to note here that the density of receptors (for both EBV strains) on B lymphocytes of these seronegative donors is comparable to that of Molt-4 cells (Table 1). Interestingly, Molt-4 cells showed the lowest EBV-R density which was 21 and 6 times lower than that of Raji cells for FITC-PEBV and FITC-BEBV, respectively (Table 1).

219

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Fig. 4. FACS profile of EBV binding to peripheral B lymphocytes from an EBV-seronegative donor. Top: background fluorescence was determined with B lymphocytes sham-treated with PBS; middle: B lymphocytes incubated with FITC-PEBV; bottom: B lymphocytes incubated with FITC-BEBV. Similar profiles were obtained with B cell preparations from three other donors (one EBV-seronegative and two

EBV-seropositive).

Further, it was found reproducibly, in two repeated experiments, that the B lymphocytes from both types of donors consisted of two different sub-populations in regard to their ability to bind FITC-EBV. Representative results illustrating this observation are shown in Fig. 4. It is noteworthy that the percentage of each of these two B lymphocyte sub-populations in regard to their ability to bind FITC-EBV varied among the 4 donors tested (2 EBV seropositive and 2 EBV seronegative), suggesting that these differences were related neither to the virus strain used, nor to the EBV antibody status of the donor.

220

TABLE 2

COMPETITIVE BINDING OF BOTH PEBV AND BEBV TO RAJI AND MOLT-4 CELLS

Cell First Second % positive cells line incubation incubation (W inhibition) a

Raji

Molt-4

_ _

_ FITC-PEBV

_ FITC-BEBV

PEBV FITC-BEBV

BEBV FITC-PEBV

PEBV FITC-PEBV

BEBV FITC BEBV

5.5

93.0

94.4

37.8 (60.0)

42.6 (54.2)

44.5 (52.1)

29.8 (68.4)

_ _

PEBV

BEBV

PEBV

BEBV

FITC-PEBV

FITC-BEBV

FITC-BEBV

FITC-PEBV

FITC-PEBV

FITC-BEBV

5.5

82.5

99.0

93.5 (5.6)

30.3 (63.3)

48.5 (41.2)

60.1 (39.3)

a The values given in brackets represent percentages of inhibition of binding of FITC-labelled EBV by

the unlabelled virus.

Characterization of the EBV receptor on Molt-4 cells

In order to learn more about the EBV-R on Molt-4 cells, we first decided to perform a competitive assay between both EBV strains. For comparative purpose, the same test was carried out. in parallel with Raji cells. Preincubation of Raji cells

TABLE 3

MEASUREMENT OF CR2 EXPRESSION ON RAJI AND MOLT-4 CELL LINES AND INHIBI-

TION OF EBV BINDING BY AN ANTI-CR2 ANTIBODY (OKB7)

Cell preparation

and treatments a

Raji sham

Raji + 0KB7 + FITC-anti-mouse IgG

Raji + FITC-BEBV

Raji + OKB7 + FITC-BEBV

Molt-4 sham

Molt-4 + OKB7 + FITC-anti-mouse IgG

Molt-4 + FITC-BEBV

Molt-4 + 0KB7 + FITC-BEBV

MFI b

21.1

207.9

724.1

84.5 (88.3)

18.4

27.9

111.4

90.5 (18.8)

% positive cells

(% inhibition) ’

4.4

99.0

98.9

4.5 (95.5)

4.5

10.0

97.0

81.8 (15.7)

a Negative controls included cells incubated with an irrelevant monoclonal antibody and cells incubated

with the anti-mouse conjugate alone.

b MFI: modal fluorescence intensity in arbitrary units.

’ The values given in brackets represent percentages of inhibition of binding of FITC-labelled EBV by

0KB7. Identical results were obtained when this assay was repeated using FITC-PEBV.

221

with either one of the two strains of EBV blocked significantly further binding of labelled-EBV of both strains (Table 2). On Molt-4 cells, however, PEBV was unable to inhibit subsequent binding of FITC-BEBV while it was able to block subsequent binding of FITC-PEBV; BEBV, on the other hand, was able to inhibit the binding of FITC-EBV from both strains. This observation suggests that there are probably two different types of receptors (or two different epitopes) involved in EBV binding to Molt-4 cells and that BEBV is able to bind to both receptor types (or epitopes) while PEBV is able to bind to only one of them (Table 2).

Nemerow and Cooper (1985) have shown that 0KB7, a B cell-specific anti-CR2 monoclonal antibody, is able to block EBV binding to Raji cells. We therefore decided to test the effect of OKB7 on EBV binding to Molt-4 cells. Results shown in Table 3 indicate that the 0KB7 monoclonal antibody does not show significant affinity for Molt-4 cells, and is thus unable to inhibit subsequent binding of FITC-EBV to these targets. When experiments were repeated using another CR& specific monoclonal antibody (i.e. antiCD21, Becton-Dickinson, Mountain View, CA), negative results were again obtained with Molt-4 cells (data not shown). This finding indicates that EBV does not use the C3d receptor/epitope, recognized by the 0KB7 monoclonal antibody, to bind to Molt-4 cells. On the other hand, the results obtained with Raji cells (Table 3) confirm that the C3d receptor (CR2) recognized by 0KB7 on these cells is also used as the EBV-R.

Discussion

This is the first study carried out to quantitate and determine, by means of FACS analysis, the expression and density of EBV receptors on various human lymphoid cell types. The data clearly demonstrate that there are significant quantitative differences in EBV-R expression among various EBV target lymphoid cell types, including between primary lymphocytes from EBV-seropositive and -seronegative donors.

After comparing the EBV-R expression in the various targets used, the EBV-R density seems to correlate with the fate of cell-bound EBV; for example, the lowest fluorescence intensity was found on Molt-4 cells in which EBV penetration does not occur following virus binding (Menezes et al., 1977; Shapiro et al., 1982); inter- mediate receptor density was found on B lymphocytes in which the virus appears to penetrate by endocytosis (Nemerow and Cooper, 1984); and the highest EBV-R density was observed on Raji cells in which EBV enters by envelope-cell membrane fusion (Menezes et al., 1977; Seigneurin et al., 1977). These observations emphasize, therefore, the importance of investigating further early virus-cell interactions at the cell membrane level in order to assess better the importance of virus receptor density as one of the possible factors dete rmining the fate of the virus particles bound to the target cell surface. In this context, it is noteworthy for example, that experiments with Sendai virus have indicated that a critical density of the virus spikes is required for cell fusion (Hosaka and Shim&u, 1977); it is thus conceivable that if a critical density of cell membrane receptors is lacking, the bound virus

222

envelope will not fuse with the target cell membrane. However, at a certain intermediate receptor density, which may be insufficient for cell membrane-virus

envelope fusion, the bound virus may still be able to penetrate, e.g. by receptor- mediated endocytosis such as has been reported for EBV infection of primary

human B lymphocytes (Nemerow and Cooper, 1984). It is unclear, however, whether

these authors used B lymphocytes from EBV-seropositive or -seronegative donors). At a low receptor density, a situation such as that observed with Molt-4 cells and B lymphocytes from EBV-seronegative donors, penetration of the bound BV may not occur (Menezes et al., 1977). Further studies are required to shed light on the more precise role played by virus receptor density in EBV penetration.

The experiments about the interaction between EBV and peripheral B lympho- cytes also demonstrate the presence of two sub-populations of B cells which bind

EBV in a differential manner. These sub-populations could represent asynchronous cells synthesizing EBV-R at different rates (e.g. cells in early G, and late G,/M phases have a higher EBV-R density and synthesis than cells in late G, and early G, phases (Wells et al., 1981)). Difference in B lymphocyte maturation stages might

also explain the observed differences in EBV binding ability among B cells (Tedder

et al., 1984). Since the B lymphocytes consisted of two sub-populations with different EBV-R

density, it would be interesting to sort out these two sub-populations using FACS and FITC-EBV; this would allow one to determine, in addition to their phenotypic

features, whether the sub-population having a lower EBV-R density would exhibit a different mode of EBV penetration or a lower susceptibility to EBV transformation and antigen expression than the one with higher EBV binding activity.

The finding of a three times lower EBV-R density on B lymphocytes of EBV- seronegative individuals as compared to B cells from EBV-seropositive donors tested (Table 1) also indicates the possibility of a differential expression of EBV-R in different groups of individuals. A similar observation using a less sensitive

method but showing a relative lack of EBV receptors on B lymphocytes from EBV-seronegative individuals was reported earlier (Gervais et al., 1981). This differential expression might be genetically determined and it is probable that individuals with low EBV-R density might resist EBV infection and those with high

EBV-R density be more susceptible to EBV. In any event, it will be important to confirm these observations with lymphocytes from a larger sample of EBV-seroposi- tive and -seronegative individuals. It is noteworthy that lymphocytes of some

patients with common varied agammaglobulinemia also appear to have a decreased EBV-R expression (Schwaber et al., 1980); while those of preleukemia patients lack

EBV-R (Anderson et al., 1983). The present results also confirm our previous observation that the EBV fluo-

resceination step does not alter the specificity of EBV binding to its target cells (Khelifa and Menezes, 1982b), since viral preparations of both strains bound to EBV-R-positive Raji cells but not to EBV-R-negative 1301 cells. Under the experi- mental conditions used, virus binding to Raji cells was found to be equal for both EBV strains, PEBV and BEBV, thus indicating that the viral preparations used were comparable. It therefore appears reasonable to consider that the differential binding

223

of these two strains of EBV to Molt-4 cells was not due to differences in EBV concentration in the virus preparations used; rather, it suggests that there may be two types of EBV receptors or two different epitopes involved in EBV binding to these T cells, PEBV recognizing only one of these and BEBV recognizing both. This consideration is further supported by the finding that PEBV is unable to inhibit subsequent attachment of FITC-BEBV on Molt-4 cells while it is able to inhibit the binding of FITC-PEBV. Another possibility, although unlikely, is that the Molt-4 receptor is expressed in a modified form so that it recognizes more easily the BEBV attachment protein than that of PEBV. This possibility remains to be investigated. On the other hand, Raji cells do not show any such differential virus strain-related binding activity. Furthermore, the results obtained with the OKB7 monoclonal antibody indicate that the EBV-R present on Molt-4 cells, in addition to showing a differential binding activity for the two EBV strains, does not utilize the C3d receptor/epitope as the EBV binding site on these T cells.

Finally, the specificity of the interaction between FITC-EBV and the various cell types used was further demonstrated by the inhibitory effect of the neutralizing monoclonal antibody 72Al on EBV binding to its targets. This antibody is known to recognize a 250 kDa viral glycoprotein (Hoffmann et al., 1980) which would thus appear to bear the virus attachment protein. Interestingly, this antibody was the only monoclonal antibody which was found to inhibit EBV binding, among 9 different EBV-MA specific antibodies tested in this laboratory (Stocco and Menezes, submitted for publication).

Acknowledgements

These results were presented in part at the UCLA Symposium on Viruses and Human Cancer, Park City, Utah, 1986, Abstract no. D118, J. Cell. Biochem., Suppl. lOA, p. 220.

We are indebted to Dr. Gerry Price for his help with FACS and to Dr. Gary Hoffmann for 72Al monoclonal antibody samples. We thank Drs. R. Khelifa and G. Ahroneim for their critical comments and suggestions. This investigation was supported by the Medical Research Council of Canada (MRCC). R.S. has a research studentship from the MRCC.

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(Received 1 August 1987; revision received 7 June 1988)