J. gen. Virol. (1983), 64, 539-547. Printed in Great Britain
539
Key words: EBV/lymphocytes/EBNA
Can Epstein-Barr Virus Infect and Transform All the B-Lymphocytes of
Human Cord Blood?
By M . Z E R B I N I ' ~ AND I. E R N B E R G *
Department o f Tumor Biology, Karolinska Institutet, S-104 01 Stockholm, Sweden
(Accepted 26 October 1982)
SUMMARY
Quantitative aspects of Epstein-Barr virus infection and transformation of human
neonatal B-lymphocytes have been investigated. 72 to 90% B-cells were obtained with
enrichment. Of the B-cells, 19 to 97% showed nuclear antigen (EBNA) 2 days after
infection. A difference between different B-cell donors in susceptibility to infection
was noted. Analysis of the virus dose-response curves obtained with twofold virus
dilutions showed that one virus particle is sufficient to induce EBNA in a cell. Of the
infected cells, 50 to 95% multiplied in microtitre wells containing a human fibroblast
feeder layer, while only a small proportion established growing colonies in soft agarose,
that could be picked up and subcultured.
INTRODUCTION
Human and some primate B-lymphocytes are the only known target cells for Epstein-Barr
virus (EBV) infection in vitro (Einhorn et al., 1978; Henderson et al., 1977). Katsuki et al. (1977)
and Steele et al. (1977) have shown that it is most likely the IgM-bearing B-lymphocytes that are
the target cells in human umbilical cord blood while IgM-, IgG- and IgA-bearing Blymphocytes may be infected in adult peripheral blood (Steele et al., 1977). The infection leads to
the establishment of polyclonal, continuously growing B-lymphoblastoid cell lines (Chang &
Golden, 1971; Gerber et al., 1969; Henle et al., 1967; Miller et al., 1971; Pope et al., 1968;
Schneider & zur Hausen, 1975). Several assays have previously been utilized to follow and
quantify EBV-directed transformation (immortalization): stimulation of D N A synthesis,
outgrowth of cells, growth on feeder layer and growth in soft agarose after infection (Chang et
al., 1976; Henderson et al., 1977; Katsuki & Hinuma, 1976; Mizuno et al., 1976; Robinson &
Miller, 1975; Sugden & Mark, 1977; Yamamoto & Hinuma, 1976). In such studies, between 0.1
and 10 % of the target cells was estimated to be transformed. Henderson et al. (1977) and Sugden
& Mark (1977) found that the transformation of target cells followed a linear response with
dilution of the virus, and this response could most easily be aligned with a one-hit curve,
indicating that one virus particle is sufficient to transform a cell.
The purpose of our study was to relate the infectibility of human cord blood B-lymphocytes as
judged by nuclear antigen (EBNA) induction to the efficiency of growth of freshly infected cells
in two systems: in suspension and in a semi-solid medium. The EBV was concentrated to obtain
excess virus for the estimation of the number of infectible cells. The target B-lymphocytes were
purified to allow direct deduction of the number of infectible and growing cells after infection.
METHODS
Fractionation of cells. Heparinized human cord blood cells were collected, and lymphocyteswere separated by
centrifugationon Ficoll-Isopaque(B6yum, 1968).Lymphoidcells at the interphase of the gradient were collected,
washed with medium (RPMI 1640supplemented with 10% heat-inactivated foetal calf serum) and macrophages
removed by carbonyliron treatment for 30 min at 37 °C accordingto Jondal (1974). B-cellswere first enriched by
passage over a nylonwoolcolumn (Cornain et al., 1975). Cells (10 x l0 T)were incubated for 1 h at 37 °C in 5 ml
plastic syringeseach containing 0.425 g of nylon wool, pretreated and washed with 20 ml of warm tissue culture
t Present address: Institute of Microbiology,University of Bologna, Osp.S. Orsola, Via Massarenti 9, 40i38
Bologna, Italy.
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M. Z E R B I N I
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medium. Unabsorbed cells were washed out with warm culture medium; adherent cells (B-enriched cell
population) were removed by incubating the nylon wool in 15 ml of warm foetal calf serum for 10 rain at room
temperature. The cells were then washed and resuspended in culture medium. A suspension of 2.5 ~ washed sheep
erythrocytes was added at a ratio of one part to five parts of cells, the mixture was layered on a Ficoll-Isopaque
gradient and centrifuged for 20 min at 2000 rev/min. The interphase was collected and the percentage of B-cells
was estimated.
Surface markers. The proportion of the ceils possessing surface immunoglobulin was determined by staining
with fiuorescein isothiocyanate (FITC)qabelled potyvalent goat anti-human immunoglobulins. Fluorescencepositive and -negative cells were counted in a Leitz Orthoplan fluorescence microscope at × 640; 100 to 200 cells
were counted in each sample.
Preparation of EBV and infection. An EBV-producing marmoset cell line B95-8 (Miller & Lipman, 1973) was
cultured in RPMI 1640 medium supplemented with 10~ foetal calf serum, 100rtg/ml streptomycin and
100 units/ml of penicillin in a humid incubator at 37 °C with 5 ~ CO2. One-week-old culture medium was collected
and the cells were removed by centrifugation at 3000 rev/min for 30 min. The supernatant was collected and
centrifuged at 10000 g for 120 min and the pellet was resuspended in 0-15 M-phosphate-buffered saline pH 7.4 to
provide 50-fold concentrated virus. The virus concentrate was passed through a Millipore filter with 0.45 Ixm pore
size and stored at 4 °C for no longer than 15 days. During this period no decrease in the infectious activity of the
virus preparation was noticeable.
One ml of appropriate virus dilution was used to infect replicate tubes each with 106 cells. The mixture was
incubated with the cells for 1 h at 37 °C. After virus adsorption, the cells were centrifuged for 10 min at
2000 rev/min. The supernatant was then removed and the cells resuspended in 1 ml culture medium.
Forty-eight h after infection, the cells were tested for EBV-associated nuclear antigen (EBNA), and processed
for the growth assay in micro-wells and the clonal transforming assay in soft agar.
Immunofluorescence. Cell smears were prepared and stained for EBNA according to Reedman & Klein (1973),
by the anticomplementary immunofluorescence (ACIF) method. At least 500 cells were counted in each sample,
differentiating between fluorescence-positiveand -negative cells. A Leitz Orthoplan fluorescence microscope was
used at x 640. The staining also labels complement receptors (Bianco et al., 1970; Einhorn et al., 1978; Yefenof et
al., 1976), which are present on all or most of the B-cells. Immunofluorescent labelling of complement receptor
differs both in location and morphology from that due to EBNA. We checked the reproducibility of the counting
of the EBNA smears by re-counting the same slide several times, and the standard deviation of the counting error
was + 4.5~ at high EBNA induction and + 1.4~ at low induction.
Preparation of feeder layer. Subcultured human foetal lung fibroblasts (after approximately 10 passages) were
collected in RPMI 1640 with 10~ heat-inactivated foetal calf serum. A 0-2 ml amount of an appropriate cell
suspension was plated in fiat-bottom micro-wells (Falcon, Microtest II) and 2 ml in Petri dishes (35 mm, Falcon
Plastics). Fibroblasts were allowed to grow as monolayers, and when saturation density was reached the cells were
irradiated with 6000 rad (Siemens roentgen unit, 220 kV, 15 mA, 1 mm Al-filter).
Growth assay in micro-wells by limiting cell dilution. The B-enriched cell populations were infected with undiluted
B95-8 virus concentrate and incubated in plastic tubes (17 x 100 mm, Falcon) for 48 h at 37 °C in a humidified
5 ~ CO2-enriched atmosphere. Infected cells diluted in warm medium to 100, 10 or 1 cell per 0.2 ml were then
added to fibroblast feeder layers in micro-wells. An identical volume of the infected and appropriately diluted cell
suspension was added. Half of the medium in each well was carefully removed every 5 days, and fresh medium
added. The plates with infected cells were kept for at least 15 days at 37 °C in humidified air in a CO2 incubator,
and each well was screened under an inverted microscope for cell growth. In each experiment uninfected B-cells
were included as control cultures.
Clonalgrowth assay in soft agarose. Confluent feeder layers of human lung fibroblasts were grown in Petri dishes
and irradiated, and l ml 0-45~ agarose medium was added at 40 °C (Sea Plaque agarose: Biochemical Division,
Maine Colloid Corp.). Subsequently, the dishes were allowed to stand for 2 h at room temperature and then for 24 h
in the humidified CO2 incubator at 37 °C. B-enriched cells (2.5 x 104) infected with twofold dilutions of
concentrated B95-8 virus were suspended in 1 ml of 0.35~ agarose medium and poured onto the first layer of
agarose. The cell cultures were kept for 1 week at 37 °C in a humidified CO 2 incubator. The number of colonies
growing in agarose.was counted under an inverted microscope at low magnification. This number is expressed as
the mean of three plates for each virus dilution. A colony was considered positive when it contained at least 25
cells.
Cloning of cells. Single clones were picked with silicone-treated capillaries under an inverted microscope. The
isolated clones were then grown in 0.4 ml of medium (RPMI 1640 + 20~ heat-inactivated foetal calf serum) in
plastic tubes (12 x 75 mm, Falcon) and incubated in a humidified CO2 incubator at 37 °C. The cultured clones
were re-fed every 5 days after gently removing half of the supernatant. Growing clones were transferred after 15
days to 17 x 100 mm plastic tubes and fed with 1 ml culture medium. Subsequently, they were transferred to
25 cm z tissue culture flasks (Falcon) at a cell concentration of 3 x 105 cells/ml.
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EBV infection of human peripheral B-cells
541
Table 1. Frequency of B-cells, EBNA-positive cells and calculated percentage of EBNA-positive
cells from the B-cell population in different experiments
Virus
preparation
A
B
C
C
D
D
E
F
G
H
Donor of
lymphocytes
I
II
III
IV
IV
v
V
VI
VII
VIII
% Bceils
90
83
80
72
72
90
90
78
72
82
% EBNA-positive
cells
61
56
63
13
24
55
87
53
25
27
EBNA-positive cells
from the B cells
Symbolof curve
(calculated)
in Fig. 1
67
79
79
19
O
33
O
63
•
97
•
68
35
32
RESULTS
Response of enriched B-lymphocytes to EBV infection
B-lymphocytes from cord blood from eight different donors were tested for EBNA induction
after infection with active, concentrated B95-8 virus. The percentage of B-cells varied between
72 and 909/o (Table 1), as estimated by surface immunoglobulin staining with FITC-conjugated
anti-human immunoglobulin antibodies. The proportion of EBNA-positive cells among the Bcells varied between 19-4 and 96.6~o (arithmetic mean: 56.5 ~).
After twofold dilutions of the virus, titration curves were Obtained as shown in Fig. 1. At high
virus concentrations there was little or no decrease of the EBNA induction response, while this
dropped rapidly with further dilution of the virus.
Theoretically, it is possible to deduce the minimum number of virus particles required to
infect a cell from the slope of the titration curves. In Fig. 2, two experiments are shown, where
the lymphocytes were infected in triplicates with different twofold virus dilutions.The titration
curves are compared with theoretical one- and two-hit kinetic curves, calculated by adopting the
analysis of the number of virus particles required to induce a plaque in lytic virus-hosl cell
systems (Davis et al., 1968). One EBNA-positive cell then corresponds to one infectious unit of
the virus preparation, like one virus-induced plaque would be in a virus-plaque assay. This
assumption is valid, since the EBNA-positive cells have not yet started to proliferate at the time
of harvest. Another prerequisite for the comparison is that the target cells are in excess
compared to the number of infectious units, and thus the analysis is only valid for lower virus
concentrations. The theoretical one-hit curve includes all virus dilution points, but the two-hit or
multi-hit (not shown) curves include only three or fewer points. This indicates that one virus
particle is sufficient to induce EBNA in a target B-lymphocyte. A similar analysis has been
performed on the titration curves in Fig. 1 at the lower virus concentrations, but the theoretical
hit curves are not shown in the figure. All these five titrations also show a dose-response
compatible with the hypothesis of one-hit kinetics.
In two experiments the same virus preparation was tested on two different donors (Table 1
and Fig. 1). A difference in EBNA induction was noted between the donors (19 to 7 9 ~ ; 33 to
639/oo).
Ability of infected cells to grow in suspension
Infected B-cells were seeded on a feeder layer of irradiated human foetal lung fibroblasts in
microplates. The microwells were prepared with 100, 10 or 1 cell(s)/well. After 15 days, the wells
were surveyed for growth. The cells were observed as clumps or flakes composed of round cells,
formed with an increase in cell number (Fig. 3a, b). The results are summarized in Table 2. The
number of wells showing growth is related to the number of EBNA-positive cells. In Table 2, the
expected nuniber of growing cells was calculated according to the Poisson distribution of
EBNA-positive cells in the wells. There was good agreement between the expected and the
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542
M. Z E R B I N I
AND
I. E R N B E R G
lf,,~
50L
I
I
\
\
\
\
\
\
o~
10
-,=
\
O
O
<
Z
<
Z
t~
\
5
\
m
\\
1
C.
0.5 ~
3
4
5
6
7
0
1
2
3
4
5
6
--log 2 Virus dilution
-log 2Virus dilution
Fig. 1
Fig. 2
Fig. 1. PercentageofEBNA-positive cellsasa functionofvirusdilution.B-lymphocytesenriched from
human umbilical cord blood were infected with twofold dilutions of concentrated B95-8 virus. These
titrations were performed in five experiments from which data are also included in Table 1, where the
symbols of this figure are provided. Two pairs of curves originate from experiments with the same
donor, but with different virus batches (C) and V).
0
1
2
Fig. 2. Percentage of EBNA-positive ceils as a function of virus dilution at lower multiplicities of
infection. Virus titration on two different donors is shown. The-plotted results are the means of
triplicate cultures for each virus dilution. SDis indicated by vertical bars. Theoretical virus-hit kinetics
curves were calculated by adopting the analysis of one-hit ( - - ) , two-hit ( - - - ) and multi-hit (not
shown) kinetics for virus titration by the plaque method (Davis et al., 1968). For this purpose, one
EBNA-positive is considered equivalent to one virus-induced plaque, i.e. both are the result of infection
with one infectious unit of the virus preparation (as the EBNA-positive cells have not yet started
growing at the time of analysis).
oLserved n u m b e r of wells with growth: the observed n u m b e r was slightly lower than the
expected number. The results imply that more than 60 ~ of the EBNA-positive cells are able to
grow under these conditions.
Cells picked up and pooled from wells seeded with 10 or 100 cells could be propagated in
suspension culture. Such cultures contained t> 90% EBNA-positive cells. The wells seeded with
one cell contained too few cells at the end of the experiment for further subculturing. Wells
loaded with uninfected cells or infected cells but no feeder layer showed no persisting growth
during the 15 day period.
Ability of recently infected cells to grow in soft agarose
A growth assay in solt agarose was adopted to allow comparison of the n u m b e r of infected
cells (EBNA-positive) with cells growing in suspension with the frequency of growth in semisolid medium. Infected and uninfected cells were plated in soft agarose (0.35 %) 2 days after
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EBV infection of human peripheral B-cells
543
Fig. 3. Growth of infected lymphocytes on human fibroblast feeder layer (a) after seeding and (b) after
15 days of culture. Unstained, living cells photographed through an inverted microscope (Diavert,
Leitz, x 320).
Table 2. Growth of EBV-infected cells on feeder layer in micro-well system followed for 15 days
Wells with positive growth
r
% B cells*
50
% EBNA-positive
cellst
24
83
51
52
12
90
87
Positive/
total
15/16
8/16
3/16
8/8
12/24
Observed
%
93
50
18
100
50
1
4/16
25
100
10
1
100
10
1
14/17
14/33
4/49
24/24
38/48
35/64
82
42.4
8
100
79
54
Cells/well
100
10
1
100
10
Expected
%:~
99"9
91
21
99.9
99.4
40
99.9
70
11
99.9
99.9
58
* Detected with fluorescent anti-Ig at the start of the infection (time 0).
"~Detection with ACIF 48 h after infection.
:~Calculated from the Poisson distribution, on the assumption that all EBNA-positive cells could grow, but not
the EBNA-negative.
infection. One week after plating, large colonies could be observed in plates with infected cells
(Fig. 4), while no cells were seen in those seeded with uninfected ceils.
Fig. 5 shows the number of clones initiated by different virus dilutions, counted on day 7 after
plating (day 9 after infection). The number of colonies shows a linear relationship to the virus
dose at low concentrations, while there is no such relationship at higher virus concentrations.
Between 1.1 and 1.4% of the total number of plated cells developed into colonies. Assuming
that only EBNA-positive cells were able to grow in the agarose, 1.4 to 3.7% of these cells grew
into colonies. This assumption is supported by the fact that it was possible to establish picked
colonies as continuously growing suspension cultures, and these cultures showed close to 100%
EBNA-positive cells.
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544
M. ZERBINI AND I. ERNBERG
500
200
I00
O
"6
,.Q
E
Z
20-
~
105
0
I
1
I
I
2
3
4
5
-log 2 Virus dilution
6
Fig. 5
Fig. 4
Fig. 4. One colony of infected lymphocytes in soft agarose 9 days after infection of B-lymphocytes with
B95-8 virus. Unstained, living cells photographed through an inverted microscope (Diavert, Leitz,
× 320).
Fig. 5. Number of colonies developing in soft agarose as a function of twofold virus dilutions of
concentrated B95-8 virus. 2.5 x 104 enriched B-lymphocytes from human cord blood were added to
each plate [82~ (O), 78~ (©) and 61 ~ CA) B-lymphocytes]. The mean + SD of triplicate plates is
shown.
DISCUSSION
EBNA induction after B-lymphocyte infection was registered on day 2 after infection. This
time point was chosen for optimal induction of EBNA prior to the onset of growth stimulation.
EBNA appears 10 to 25 h after the infection of lymphocytes, followed by the stimulation of
cellular DNA synthesis at 40 h (Aya & Osato, 1974; Einhorn & Ernberg, 1978; Menezes et al.,
1976), mitosis at 48 h (I. Ernberg, unpublished observation) and, subsequently, the first cell
division. Thus, the frequency of EBNA-positive cells on day 2 reflects the number of initially
infected cells before their number is changed by cell division.
Infection with different virus dilutions showed a plateau of EBNA induction at high virus
concentrations. This implies that all susceptible cells in the target population were infected.
Between 13 and 87 ~ of the whole lymphocyte population was infected. Only B-lymphocytes are
susceptible to EBV-infection (Einhorn et al., 1978; J_ondal & Klein, 1973). Thus, taking into
account the small variable contamination of non-B-lymphocytes, between 19 and 97 ~o of the Bcells were infected. In six out of ten experiments, more than half of the B-ceils were infected.
Considering that almost all susceptible cells were infected, and that there was no influence on
the number of infected cells due to cell proliferation, we conclude that usually the majority of the
B-cells in human cord blood may be infected. Moss et al. (1981) have reported a similar situation
with adult B-lymphocytes. When they studied the appearance of EBV-associated cell surface
antigen (LYDMA) in relation to the appearence of EBNA after infection of adult human Blymphocytes, they found a high level of EBNA induction. In experiments where they blocked Bcell proliferation with 10 mM-thymidine, they still detected 70 to 7 6 ~ EBNA-positive cells after
infection.
We cannot exclude the possibility that there may be a resistant subclass of B-lymphocytes.
Actually, Tsukuda et al. (1982) have recently demonstrated, in this laboratory, that by
transplanting EBV receptors to purified B-lymphocytes, they could enhance virus-induced
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EBV infection of human peripheral B-cells
545
Table 3. Summary of infectibility and growth in micro-wells and agarose, from ten different
experiments
Frequency of B-cells
Frequency of EBNA-positive cells
Frequency of EBNA-positive cells/B-cells
Frequency of growth on feeder layer/EBNA-positive cells
Frequency of growth in agarose/EBNA-positivecells
Percentage
72-90
13-87
19-97
50-95
1.4-3.7
immunoglobulin synthesis in these cells after EBV infection. The receptors were transferred by
membrane vesicles from EBV receptor-positive cell lines. Moreover, they observed a relatively
larger proportion of IgG-producing cells, compared to IgM-producing cells, in the receptor
transplanted population. One possible interpretation of this finding is that IgG-bearing Blymphocytes partly lack, or have a low concentration of, EBV receptors, and thus would be more
difficult to infect than IgM-positive B-lymphocytes.
The variation observed as between donors may be ascribed to variable conditions in the target
ceils, to a difference in.susceptibility between B-cell subpopulations, and to variations between
virus batches. The latter possibility is unlikely in the experiments where the response reached
plateau level with concentrated virus. However, two virus batches tested against the ceils of the
same donor resulted in different levels of EBNA induction. The difference found in sensitivity
to infection between different donors in two experiments is interesting. Unfortunately, material
derived from fresh human cord blood does not allow repeated experiments on the lymphocytes
from the same donor. Thus, the possibility of different susceptibility of different donors has to be
further investigated in purified B cells from adults.
The relation between virus dose and EBNA induction is close to being linear at low virus
concentrations. When the target cells are in excess, conclusions may be drawn about the slope of
the dose-response curve. If the experimental error is taken into account this curve is most easily
aligned with a one-hit response. Thus, one virus particle is enough to induce EBNA in a B-cell.
Other authors have shown that virus-induced growth in semi-solid medium and in suspension
also follows an ideal one-hit curve on virus titration (Henderson et al., 1977; Sugden & Mark,
1977).
The efficiency of the growth of EBV-infected cells on feeder layers has been reported to be
high (Henderson et al., 1977). In this paper we report an outgrowth frequency of 50 to 95 ~o of the
EBNA-positive cells in such a system. The efficiency of outgrowth after plating on average one
cell/well in micro-plates, is remarkable (63 to 9 3 ~ of the expected, assuming that all EBNApositive cells had been growing). From micro-wells with initially 10 or 100 cells plated, the cells
could be isolated and further subcultured. We have also managed to subculture cells from wells
with one cell originally plated. However, this requires 6 to 8 weeks proliferation in the original
micro-plate until the cell number is sufficient. For technical reasons the number of clones
isolated in this way is low (I. Ernberg, unpublished results).
Nilsson et al. (1977) have shown that growth in soft agarose is related to tumourigenicity
among EBV-carrying cell lines. Colony formation in soft agarose has also been used to monitor
EBV-induced transformation (Mizuno et al., 1976; Sugden & Mark, 1977). It was therefore of
interest to see whether freshly infected B-lymphocytes could develop clones in agarose, and if so,
how this was related to the primary infection of these cells as measured by E B N A induction. The
results show that a smaller population of cells, compared with that of the cells growing in
suspension, is able to grow in soft agarose: 1.4 to 3-7 ~o of the EBNA°positive cells. The colonies
formed could be picked and subcultured. The developing cell lines were EBNA-positive.
The results are summarized in Table 3 : a large proportion of cord B-lymphocytes may be
infected and a majority of the infected cells start to grow in suspension. However, only a small
fraction is able to grow in semi-solid medium.
This work was supported in part by Contract No. NO1 CP 33316 within the Virus Cancer Program of the
National Cancer Institute, the Swedish Cancer Societyand the King Gustaf V Jubilee Fund. M.Z. was supported
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546
M. Z E R B I N I AND I. E R N B E R G
by a grant from C.N.R. Roma, Progetto Finalizzato Virus, Contracts No. 76. 00672.84 - No. 77.00281.84, and
I.E. by a fellowship in Cancer Immunology from the New York Cancer Research Institute.
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