A microassay for quantitatively detecting the epstein-barr virus receptor on single cells utilizing flow cytometry

Journal of VirologicalMethods,

3 (1981)

Elsevier/North-Holland

Biomedical

A

FOR

MICROASSAY

127

121-136

Press

QUANTITATIVELY

DETECTING

THE

EPSTEIN-BARR

VIRUS RECEPTOR ON SINGLE CELLS UTILIZING FLOW CYTOMETRY

ALAN WELLS’,

HARALD

STEEN’,

SEM SAELAND2,

TORE GODAL

and GEORGE

KLEIN’

IDepartment

of Tumor Biology, Karolinska Instituter, S IO4 01 Stockholm, Sweden; and ‘Biophysics

Department,

and 31,aboratory for Immunology

Department

Society, Norsk Hydro S Institute for Cancer Research, (Accepted

24 March

A quantitative utilizing B95-8

was assessed methods. enabling

and Norwegian

Cancer

1981)

microassay

fluorescein-conjugated and P3HR-1

of Pathology

The Norwegian Radium Hospital, Oslo. Norway

strains

virions were labelled

by flow, cytometry,

Cell size and cellular us to calculate

for detecting

the Epstein-Barr

The

and adsorbed

to a variety

the results DNA content

EBVR density

and analyzing

is presented.

being directly

test

receptor

virus Relative

binding

with those obtained

simultaneously synthesis

receptor

substrain-specific.

of targets.

comparable

were measured

and to correlate

is virus

(EBVR) Both

the

of virus by earlier

with virus binding,

thus

and cell cycle stage.

INTRODUCTION

Recent advances in microinjection of the Epstein-Barr virus (EBV) (Miller et al., 1979; Graessman et al., 1980) have demonstrated the role of the EBV receptor (EBVR) as the controlling element in EBV infection. Bypassing the membrane barrier, a few groups have succeeded in producing both lytic and transformation cycles with EBV in a wide range of naturally non-permissive systems (Volsky et al., 1980; Shapiro et al., 1981). As EBV has been strongly associated with three human diseases: infectious

mono-

nucleosis, African Burkitt lymphoma, and undifferentiated nasopharyngeal carcinoma (for review, see Epstein and Achong, 1979), elucidation of the EBVR is also important clinically. Earlier investigations of the EBVR have relied upon indirect assay systems to observe and characterize the receptor (Sairenji and Hinuma, 1973; Yefenof et al., 1976; Menezes et al., 1977). The accepted viral absorption bioassay, developed by Sairenji and Hinuma (1973) is a time-consuming, indirect test which is only semi-quantitative. The alternative method, indirect immunofluorescence (IMF), suffers from many deficiencies, the major one being the lack of a clearly defined anti-EB virion serum. As such, both systems offer limited information. To overcome these deficits in studying EBVR, we recently developed a direct radiolabelled virion binding assay (.Koide et al., 1981). This method is quick, simple and quantitative, offering the opportunity to study a number or EBV-related functions (Wells et al., 198 1). It requires mass quantities of cells, however, being incapable 0166-0934/81/0000-0000/$02.50

@ Elsevier/North-Holland

Biomedical

Press

128

of yielding information

on single cells. Here we report a method which allows the EBVR

to be observed at the single cell level. Statistically significant numbers of cells are analyzed by flow cytometry in terms of EBVR and a second parameter which may be cell size or cellular DNA content, the latter indicating the position of the cell in the cell cycle. Thus, subpopulations of cells, in regard to one or more of these parameters, readily distinguished and quantitated separately. MATERIALS

may be

AND METHODS

Cells Raji (Epstein

et al., 1966) and Daudi (Klein et al., 1978) are EBV-carrying

Burkitt

lymphoma (BL)-derived lines. Ramos (Klein et al., 1975) is an EBV-negative American BL cell line. YAC (Klein et at., 1979) is an EBVR-negative line derived from murine T cells. 138/79 and 282/79 are biopsy material taken from patients with lymphoplasmacytoid lymphomas of B-cell origin. 138 represents leukaemic cells isolated from peripheral blood by Ficoll-Isopaque centrifugation. 91% of the cells were B cells staining monoclonally for &M/u. Cell suspension 282 was lymphoma cells, of which 62% of the cells were B cells staining monoclonally for IgM/u. The methods for preparing the cells and their histopathological and immunological characterization have been described elsewhere (Godal et al., 198 1; Laudaas et al., 1981). The biopsy material was stored on liquid nitrogen until used. Cell lines were grown as suspension cultures in RPM1 1640 medium (Gibco, Scotland) supplemented with 10% fetal calf serum, streptomycin (100 pg/ml), and penicillin (200 units/ml), at 37”C, 5% COz and 60% humidity. Virus EBV substrams B95-8 and P3HR-1 (B and P virus, respectively) were utilized to ascertain the presence of the EBVR. B-virus producer B95-8 cells and P-virus producer P3HR-1 cells (Klein et al., 1978) were grown as suspension cultures for one week at confluence

before the virus was harvested.

The virus was then concentrated

and purified

over lOOO-fold as previously described (Dolynuik et al., 1976; Koide et al., 1981). Briefly, virus-containing, cell-free medium was centrifuged at 13,OOOg for 2 h to pellet the virus. The pellets were resuspended in 0.5 mM phosphate buffer, pH 8.0, with 1 mM EDTA and 0.02% NaNa, to yield a 500-fold concentration. This was then homogenized with a tightfitting douncer and clarified by centrifugation at 9000 g for 5 min. The supernatant was then either labelled directly or purified on consecutive 5-3% and lo-30% dextran gradients. The gradients were run under identical conditions; 21,000 r.p.m. for 1 h in a Beckman (U.S.A.) SW27.1 rotor. In both gradients the radioactivity and viral infectivity peaks coincided at 17-18% dextran (Koide et al., 1981). This fraction was collected for use. The non-purified virus was stored at -90°C and the dextran gradient virus at 4°C. Viral infectivity was ascertained by the induction of the EBV-associated antigens, EBNA (Reedman and Klein, 1973) and EA (Klein et al., 1971) in indicator cells.

129

FITC labelling of virus Fluorescein

isothiocyanate

(FITC) was directly linked to EBV virions using previously

described methods (Johnson et al., 1978). The dye/protein ratio was 20 1.18dye/mg protein, at a protein concentration of approximately 20 mg/ml as determined by the Lowry method (Lowry et al., 195 1). The conjugation was performed at room temperature for 2.5 h. A Sephadex G-25-M t,Pharmacia, Sweden) column, followed by overnight dialysis at 4”C, was empioyed to separate the free dye from the conjugates. The conjugates were stored at -90°C. Rhodamine labelling of virus EB virion were labelled a fluorescent red by conjugation with tetramethylrhodamine isothiocyanate, in a manner similar to the FITC labelling (Johnson et al., 1978). The conjugates of this dye were treated identically to the above. Virus-binding assay The viral-binding assay was performed as described with minor modifications (Koide et al., 1981). One million cells from the lines or 1.5 X lo6 cells from the biopsy material were centrifuged at 600 g for 7 min. 100 /.d of fluorescent virus diluted in phosphatebuffered saline (PBS), representing approximately lo8 virions per cell, were added to the cell pellet. This was then incubated for 30 min at room temperature with gentle shaking. The cells were washed three times in PBS and stored on ice for less than 3 h for cytometric analysis. Viability monitoring of the cells showed no significant cell mortality during the procedure. Propidium iodide labelling After binding virus, the cells were stained for DNA content propidium iodide (Johnson et al., 1978). The cells were washed free of unbound virus, pelleted, and resuspended in 500 ~1 0.1 M Tris-HCl buffer, pH 7.6, containing 0.4% ribonuclease (DNase-free) and 25 pg propidium iodide. This was incubated on ice for between 10 min and 3 h before analysis. Flow cytometry The flow cytometric analysis was performed with a microscope-based flow cytometer (Steen and Lindmo, 1979; Steen, 1980) comprised of a 5 W Ar- laser, operated at 476 nm, as the excitation light source. The FITC fluorescence was isolated by an interference filter transmitting between 500 and 525 nm, while propidium iodide and rhodamine fluorescence were detected through a filter transmitting above 620 nm. Between 50 and 100 X 1O3 cells were measured from each sample, at a rate of approximately 500 cells/set.

130

RESULTS

Comparison of FITC-virus binding with previous binding methods Table 1 compares the results of EBV binding to three cell lines, Raji, Daudi and Ramos, obtained with three different assay systems. All parameters are adjusted to Raji. To calculate the relative fluorescence of the lines, the values of the peak channel were used. The values obtained by the flow cytometer are within two standard deviations of the radio-binding

assay. However, as the fluorescence

scale is compressed

so the complete

spectrum is contained on the screen, minor shifts in the peak position are magnified in calculations. A second correlation between Daudi and Raji, utilizing the percent of cells within the peak channels (channels containing more than 0.5% of the cells) and the fluorescent intensity of those channels, radio-binding value of 78.

presents a ratio of 79, which is quite analogous to the

Binding of B virus and P virus Both viruses were FITC-labelled and bound to the cell lines and the biopsy material (Figs. 1 and 2). All targets showed similar profiles for both viruses. In the case of the cell lines, the peak channels for both viruses are identical (Table 2). Additionally for Raji and Daudi, the peak fields were analyzed, the percent of cells counted in each being comparable. These results support our previous findings that Raji, Daudi, and Ramos contain only the dual-virus-recognizing EBVR (Wells et al., 1981). The biopsy material,

TABLE

1

Comparison Cell line

of EBV-binding Relative binding

Raji

levels obtained

fluorescenta

61

Ramosf

58

Radio-bindingb

level 100

100

Daudi

in three different

assay systems Virus adsorptionC

Surface area d

100

100

1.00

88

82

0.81

64 + 5

61

73

0.79

n.c. h

n.c.

0.96e og

0

Based on binding

peak channel

Wells et al. (1981) Performed

og

of FITC-B virus.

(2 S.D.).

according

to Sairenji

and Hinuma

(1973).

Based on cell size peak channel. Based on total relative Scale readjusted Defined

intensity

to scale of Raji.

as zero specific

Not calculated.

density

78 +6

78e YAC

EBVR

binding.

of peak channels.

131

Fig. 1. Binding of P and B virus to cell lines. Virus was labelled and bound to cells as described. The ordinatle increases to the right and represents FITC fluorescence; the abscissa, towards the reader and slightly to the left, is cell volume (or rhodamine fluorescence in C), and the vertical is representative of the number expended

of cells. The vertical

for Ramos

were analyzed

in three

scale is linear.

The abcissa

is constant,

(J, K, L). YAC (A, B), Raji (D, E, F), Daudi ways:

in the absence

and B virus (F, I, L). Additionally

Raji adsorbed

but the ordinate

(G, H, I), and Ramos

of virus (A, D, G, J), and adsorbing FITC-B virus and rhodamine-labelled

138 and 282, were both scored as approximately

25% EBVR-positive,

is twice (J, K, L)

P virus (B, E, H, K), P virus (C).

regardless of virus

substrain. In the negative controls, less than 3.5% of the cells were in the positive field (Tabel 2, Figs. 1 and 2, parts A, D, G, J). Two-colour analysis of virus binding (Fig. 1C) demonstrates the dual-virus receptor on Raji. B virus was FITC-labelled and is represented on the ordinate; P virus, rhodaminelabelled, is shown on the abcissa. The greater strength of the FITC-labelling procedure is reflected in the slope of the crest being greater than 1; therefore, the observed asymmetry is artifactual.

132

Fig. 2. Binding of P and B virus to biopsy material. The axes are arranged ception show

that

the vertical

138 (A-F),

axis in the second

the last two

282 (G-L).

and fourth

row is in logarithmic

The columns

are absence

as in Fig. 1, with the exscale. The first two rows

of virus (A, D, G, .I), FITC-P

virus (B, E, H, K), and FITC-B virus (C, F, I, L).

EB VR density Cell volume was ascertained by measuring the degree of light scatter caused by the target cells. Surface area of a spherical object is obtained by raising the volume to the 2/3 power. As lymphoid cells in suspension can be approximated as spheres, this relationship is valid. The ratio of relative fluorescence to relative surface area is given for the cell lines (Table 1). Two values are presented for Daudi: that derived from the peak channel, 0.8 1, and that obtained by using the peak field intensity, 0.96. If the relative radio-binding values are utilized, then the values of 0.95 and 0.87 are realized for Daudi and Ramos, respectively. Rough approximations for the biopsy materials EBVR (+) cells present values near 0.7 (data not shown).

133

TABLE

2

Binding

profiles

Target

Peak channela B virus

of B and P virus Positive

fieldb

P virus

Arbitrary

% Positive B

P

IleldC

Unlabelled

%

In

field

B

P

Raji

6,8

6,8

2-31,3-31

> 95

> 95

3.2

4413,4-13

65.9

67.8

Daudi

4,6

4,6

2-31,3-31

> 95

> 95

4.3

2-12,3-14

80.2

78.4

3.5,5

3,5

2-31,2-31

> 90

> 90

< 5.0

25

27

22

19

Ramos YAC

2-31,3-31

138

2-31,2-31

2.5

1.531,2-31

282 a

Scales adjusted

b

Region

c

See Results.

< 3 <5 3

to Raji’s.

designated

positive

for FITC-virus

bound-adjusted

to scale on Raji figure.

Relation between cell cycle and EBVR expression Propidium iodide staining of DNA was employed to distinguish the different cell cycle stages (Fig. 3). The profiles for Daudi and Raji are similar, the only difference being fluorescent intensity. The Gr phase is represented between the ordinate values of 11 and 15, inclusive, S from 16 to 21, and G, and M, from 22 to 25. For both cell lines, the EBVR-associated fluorescence increased through S and Gz to approximately 40% above the mean value in Gr, indicating that EBVR synthesis takes place during the entire cell cycle, though primarily in Gr .48% of Daudi and 59% ofRaji were observed to contain the DNA amount which we designate as the G, phase.

Fig. 3. DNA content

versus

stained

with

propidium

amount

of DNA. A) Daudi.

virus binding.

iodide.

The

B) Raji.

The

abcissa

targets

represents

adsorbed FITC

FITC-labelled fluorescence;

P virus the ordinate

before shows

being the

134

DISCUSSION

Indirect

immunofluorescence

successfully

to study

years. Its applicability

surface

(IMF), structures

to EBV-binding

the sandwich

technique,

has been employed

on single cells in various systems

for many

studies has, however, been limited by two major

factors: 1) virion envelope components are present on producer cells which are devoid of EBVR (Wells et al., 1981); and 2) only within the past year have there been clearly defined monospecific and monoclonal antibodies against the virion components (Hoffman et al., 1980; Thorley-Lawson, 1981). Unfortunately, as the different EBV substrains are qualitatively similar in envelope constituents (Qualtiere and Pearson, 1980; Edson and Thorley-Lawson, 1981; Koide et al., 1981), the newly developed monoclonal antibodies are incapable of distinguishing between the strains. These are not useful, therefore, in the study of strain-specific EBV receptors present on certain unusual cell lines (Wells et al., 1981). By directly fluorescein-labelling the virus before adsorbing it to target cells, these problems are overcome. Advances in technology to measure and analyze fluorescent material,

allow objective and statistical

handling

of the data. Computer

analysis of fluor-

escent EBV binding is directly comparable to results obtained by other methods (Table 1) which require large numbers of cells. Thus, flow cytometric analysis of fluorescein-labelled EBV functions both as a single cell analyzer and as a quantitative microassay. EBV-binding levels among different cell lines show a heterogeneity of receptors per cell. Analyzing binding in terms of cell volume, it is possible to determine whether this difference is due to cell surface area or receptor density. Daudi, presenting a lower level of virus adsorption than Raji, retains the high EBVR density of the latter, as its surface area is correspondingly less (Table 1). The low level of binding of Ramos, alternately, is due to both a smaller area and lower density of EBVR. The determination of the EBVR density in biopsy material is complicated by the fact that the cell population is heterogeneous, usually with only a subpopulation being receptor-positive. Flow cytometry is capable of distinguishing these positive cells, so that they can be analyzed independently. The 25% of the cells in the 138179 and 282179 which adsorb virus show very low levels of binding, but this is due mainly to their small size as opposed to EBVR density; here they demonstrate a relatively high ratio of 0.7. In a radio-binding assay, these biopsies yield misleading values of approximately 15% of Raji’s binding due to the combination of small surface area and minority subpopulation. It is obvious that flow cytometric testing is preferable to radio-binding assaying of heterogeneous cell populations, as the former can distinguish the different subgroups and analyze them idependentlY. Additionally, we have examined the relationship of EBVR to the cell cycle as an example of two-colour, two-parameter analysis. Based on DNA content of the cells, we are capable of roughly dividing the cells into Gr (and G,), S, and G2 and M phases. EBVR was clearly present in all phases of the cell cycle. Apparently, receptor synthesis occurs during the entire cycle, although the major part occurs in Gr . According to the DNA distribution, the cells spent approximately half of the cycle in Gr , whereas roughly 70% of

135

the EBVR was synthesized

during this period. While further experiments

asses the validity of this observation,

this presents an opportunity

are necessary to

to preferentially

label

the EBVR by cell synchronization. We have presented

a technique

which can detect the Epstein-Barr

single cells and relate its presence directly to other important

virus receptor

on

parameters such as cell size

and cell cycle stage. This method is rapid, accurate, quantitative, direct, and virus substrain-specific, properties which are not present with the previously utilized IMF. ACKNOWLEDGEMENTS

This investigation was supported by Grant Number 2 RO 1 CA 14054-07A1, awarded by the National Cancer Institute, DHEW, the Swedish Cancer Society and King Gustav V Jubilee Fund and the Norwegian Cancer Society. We wish to express our sincere gratitude to Ms. Mona Hedenskog and Ms. Kari Hildrum for their excellent technical assistance and Mrs. Anita Lofgren for typing the manuscript. A.W. is a recipient of an ITT International Fellowship and a fellowship from Concern Foundation. H.S. is supported by the Norwegian Cancer Society. REFERENCES

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