J. gen. ViroL (I972), I4, 77-86
77
Printed in Great Britain
In vitro Immune Responses o f Spleen Cells from
Friend Virus Infected Mice
By B. N. D R A C O T T , N I N A W E D D E R B U R N
AND M. H. S A L A M A N
Department of Pathology, Royal College of Surgeons of England,
Lincoln's Inn Fields, London WCzA 3PN
(Accepted 14 September I97I )
SUMMARY
The primary immune response to sheep erythrocytes o f spleen cells cultured
from mice infected with Friend virus was compared with that of normal spleen cell
cultures. Significant depression of the response, as measured by the production of
haemolytic plaque-forming cells, was first observed when mice had been infected for
3 days prior to cell culture. This depression became more severe as the infection
period was prolonged.
After 4 days infection in vivo the peak of the in vitro response was reduced to a
mean of I 1% of normal values. No depression was observed during the first 48 hr
of the development of the response in such cultures.
The immune defect was shown to exist only in the non-adherent (lymphocytelike) fraction of the spleen.
INTRODUCTION
Mice infected with Friend virus (Friend, 1957) produce depressed primary and secondary
responses in vivo to sheep erythrocytes (Old et al. 196o; Odaka et al. i966; Salaman &
Wedderburn, 1966; Wedderburn & Salaman, 1968; Ceglowski & Friedman, I968; Bennett &
Steeves, 197o). The response to a number of other antigens is also depressed (Millian &
Schaeffer, 1968; Hirano et al. 1969). It would appear that the virus acts principally on the
more primitive elements of the haematopoietic system (Mirand, I967; Friedman & Ceglowski, 1968; Chan et al. 1968) but the mechanism of the immune depression is not fully understood.
We have studied the effect of Friend virus infection in vivo on the primary immune response
to sheep erythrocytes in vitro, using the spleen culture technique of Mishell & Dutton (1967).
In this system a minimum of three cell types are believed to co-operate in the production
of the response (Mosier & Coppleson, 1968; Haskill, Byrt & Marbrook, 197o; Dutton et al.
197o; Tan & Gordon, 1971). To study the effect of Friend virus on these co-operating ceils
we have used the cell-adherence separation technique of Mosier (1967).
METHODS
Animals. Two to 6-month-old Balb/c mice of either sex were used for all experiments.
Mice in any one experiment were of the same sex and differed in age by not more than
I week.
Antigen. Sheep erythrocytes (SE) from selected individual donors were obtained from
Wellcome Reagents and stored in the cold for 2 weeks before use.
6
VIR 14
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 16:04:48
78
B.N. DRACOTT, N. WEDDERBURN AND M. H. SALAMAN
Friend virus. Complete details of the preparation of Friend virus (FV) were given in a
previous paper (Wedderburn & Salaman, I968). Mice were infected intravenously with
o.I ml. doses of citrated plasma or spleen extract containing lO4 ID5o of the virus.
Media. Foetal calf sera were obtained from Flow Laboratories and from Armour Pharmaceuticals. Batches were selected for their ability to support the in vitro immune response
of spleen cells and stored at - 2 0 °. Eagle's MEM was obtained from Wellcome Reagents;
supplements from Flow Laboratories and from BDH Chemicals.
Culture medium, nutritional mixture, and balanced salt solution (BSS) were prepared as
described by Mishell & Dutton (I967), except that 4o #g./ml. L-asparagine was added to the
culture medium. Eagle-Hepes (Eagles's MEM buffered with IO mM-HEPES (BDH Chemicals)) was used to prepare the cells for culture, and to stabilize the pH of the culture medium
during cell separation and harvesting.
Cell culture. Mice were killed by cervical dislocation. Their spleens were cut into three or
four pieces and pressed gently through a fine mesh stainless-steel sieve using Eagle-Hepes
containing 30 ~ inactivated calf serum (Wellcome Reagents) as diluent. Clumps of cells
were dispersed by repeated aspiration through a Pasteur pipette; any aggregates remaining
were allowed to sediment. The overlying cell suspension was washed once in serum-free
Eagle-Hepes and resuspended in I o ml. complete culture medium per spleen. This suspension
contained 12 to 2ox IOG nucleated cells/ml, with approximately 9o ~ viability. The suspension was fractionated as described under 'Cell separation', or incubated directly; in the
latter case I ml. volumes were placed in 3o mm. plastic tissue culture plates (Sterilin) with
o'o5 ml. washed IO ~ SE in BSS. The plates were incubated at 35 ° on a platform rocking at
6 cyc./min, in a water-saturated atmosphere of IO ~ CO2, 7 ~ 02, and 83 ~ N2. Cultures
were fed daily with o-I ml. nutritional mixture.
Cell separation. Adherent macrophage-like cells (fraction M), and non-adherent lymphocyte-like cells (fraction L) were prepared by the technique of Mosier (I967), modified as
follows:
One ml. samples of cell suspension were incubated in 3o mm. culture plates without rocking or the addition of SE. After 3o min. the plates were gently agitated and placed on the
rocker for a further 3o min. incubation. The medium in each plate was then replaced with
2 ml. Eagle-Hepes and loose cells displaced by pipetting the liquid over the surface of the
plate. The resulting cell suspension was discarded and the washing procedure twice repeated,
leaving an even layer of firmly attached cells (fraction M). Microscope counts of IO random
fields/plate showed, over numerous experiments, a range from 3 to 9 x lO5 adhering cells/
plate. In any one experiment all plates contained approximately the same number of adherent cells.
Fraction L was prepared by incubating I o ml. volumes of suspension (adjusted to contain
I5 x lOGviable nucleated cells/ml.) in 9o ram. plastic tissue culture plates (Sterilin) on the
rocker for 3o min. without the addition of SE. Some 7 to 8 ml. of non-adherent cell suspension was then carefully removed from each plate and transferred to fresh 9o ram. plates,
which were rocked for a further 3o min. Each plate finally yielded 6 to 7 ml. of suspension
containing some 50 x lOGviable non-adherent cells.
These were centrifuged at 25og for 5 min. and resuspended in fresh culture medium to the
original cell concentration. Samples of I ml. were transferred either to fresh 30 mm. plates
or to plates containing fraction M cells, which, after the addition of SE, were incubated as
previously described.
Haemolytic plaque assay. Cultured cells were harvested into cold Eagle-Hepes (using a
policeman to remove adherent cells), centrifuged in the cold at 25og for 5 rain. and resus-
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 16:04:48
In vitro immune depression by Friend virus
79
pended in an appropriate volume of cold BSS. Haemolytic plaque-forming cells (PFC) were
enumerated by the technique of Jerne & Nordin (I963), modified by the use of BSS in both
agar layers.
Cell counts. Viable nucleated cells were enumerated directly in a haemocytometer, taking
the exclusion of o'o5 ~ nigrosin in Ringer's solution as evidence of viability. Cell counts were
performed on each suspension prepared for culture, and on each culture prepared for PFC
assay.
Experimental design. Groups of two to ten mice were used, depending on the number of
plates required. Two or four plates were prepared for each variable; these were harvested in
pairs and assayed in triplicate for PFC. The mean number of plaques in each set of assay
plates was expressed as PFC/Io 6 viable nucleated cells, as counted in the suspension prepared for assay.
Where differences between experimental data are claimed, they have been established as
statistically valid by means of the ' Student' t test for small samples with a selected value
for p of o'o5.
RESULTS
Primary response of infected cells
Preliminary experiments were performed to determine whether the primary response to
SE in vitro was depressed as a result of FV infection in vivo. Groups of mice were infected at
intervals ranging from I hr to 7 days before their spleen cells were cultured. These cultures,
together with control cultures from uninfected mice, were assayed for PFC against SE after
5 days incubation with SE (' stimulated'), and without SE (' unstimulated'). Unstimulated
cultures were included since PFC against SE develop in excess of background in the absence
of this antigen. This may be due to cross-reacting antigens in the foetal calf serum included
in the culture medium (Mishell & Dutton, 1967; Marbrook, ~967).
Fig. x shows that after a minimum of 3 days infection in vivo the response in vitro was
depressed, and that this depression increased as the in vivo infection period was extended.
These findings are comparable to, though not identical with, those made in vivo (Wedderburn
& Salaman, 1968). The effect of FV on unstimulated PFC levels is also shown in Fig. I ; although variations both above and below the corresponding control range were observed,
these were at most only marginally significant.
Primary responses of spleen cells from 4-day infected mice
There is evidence that cell transformation occurs as early as I day after infection in vivo
with FV (Rossi, Cudkowicz & Friend, I97O), and definite morphological and histological
changes in the spleen are detectable 4 days after infection (Metcalf, Furth & Buffet, I959;
Dawson, Fieldsteel & Bostick, I963). The spleen enlarges throughout the course of the
disease as the number of nucleated cells it contains increases. This increase is due mainly to
the abnormal proliferation of erythroid precursors. Thus it becomes progressively less
meaningful to relate the response of an infected suspension to that of a normal suspension
containing the same number of cells, since the ratio of immunocompetent cells to total cells
in the former may have been reduced by this proliferation of non-lymphoid cells. We have
observed that sub-maximal PFC levels were obtained even when normal cells were cultured at
concentrations in excess of 2 )< IoT/ml. As it was not possible to culture equivalent fractions
of normal and infected spleens without exceeding this concentration in cultures from mice
infected for more than 4 days, we have worked routinely with spleens from 4-day infected
mice. The weight and total cell number of such spleens did not exceed those of normal spleens
6L2
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 16:04:48
80
B . N . D R A C O T T , N. W E D D E R B U R N AND M. H. S A L A M A N
by m o r e than IO ~ , so that c o m p a r i s o n s between P F C counts per million viable cells m a y
still be meaningful. H e r e a f t e r the t e r m ' i n f e c t e d cells' is used to d e n o t e spleen cells f r o m
mice infected with F V 4 days p r i o r to culture.
T ab l e I gives the results o f ten representative experiments in which the 5-day p r i m a r y
responses o f n o r m a l a n d infected cultures were c o m p a r e d . A l t h o u g h there was considerable
v ari at i o n in P F C n u m b e r s between experiments, the stimulated response o f infected cultures was always m a r k e d l y depressed in relation to the c o m p a r a b l e uninfected control.
O n c e again F V only slightly affected the unstimulated response.
T h e degree o f i m m u n e depression exerted by F V was similar whether the results were expressed as P F C / I o 6 cells originally cultured, or as P F C / I o 6 cells surviving on day 5, since
I
1000 -
I
I
]
I
I
I
I
_-
~
Control
-~ 100
-~.
o_
.
.
Control
.
/
.
~ ~:~'~
~"-..
/'"
"o"
10I
I
I
I
I
I
I
I
--7
--6
--5
--4
--3
--2
--I
0
Day of FV infection
Fig. I. Effect of length of infection in vivo on subsequent primary reponse to SE in vitro. *, Viable
nucleated spleen cells after 5 days incubation. @--@, FV-infected SE-stimulated. © - - - ©, FVinfected, no SE added to cultures. Parallel lines indicate range of responses of uninfected cultures.
Tab l e I. Primary response to S E in vitro of spleen celts from normal
and 4-day FV-infected mice
PFC/m 6 viable cells assayed after 5 days culture
c
SE-stimulated
No SE to cultures
r
~
r
Expt
Normal
In~cted
Normal
In~cted
19
22
32
36
37a
37b
45
47
51
52
Geometric mean
% of normal
780
492
325
497
429
84o
726
247
848
574
536
Ioo
44
20
I2
I27
I31
42
86
55
I7o
84
59
II
63
26
23
6I
72
59
--lol
75
54
IO0
20
I8
I7
25
33
I3
--83
rl
22
4I
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 16:04:48
In vitro immune depression by Friend virus
8I
there was similar cell survival (approximately 25 %) in normal and infected cultures. However, as viable ceil counts did not distinguish between different nucleated cell types, it could
not be determined whether the ratio of immunocompetent cells to total surviving cells was
the same for both categories, ff the results were expressed as PFC/spleen, as in previous
in vivo studies (Wedderburn & Salaman, I968), the degree of immune depression was not
materially altered. For example, in Expt 35 (Table I) the normal and infected responses were
respectively 726 and 86 P F C / I o 6 cells assayed, and respectively 25 400 and 2800 PFC/spleen;
a ratio of approximately 8: I in both cases.
I
I
|
I
I
I
I
I
1000
100
%
/,
J
/ / , ~~ ,-,,.,
. ~ , -~ . . . . o . .
>
10
O-
e"
I
I
I
0
1
2
I
I
3
4
Days in culture
I
I
I
5
6
7
Fig. 2. Development of primary responses to SE in vitro of spleen cells from normal and 4-day
FV-infected mice. *, Viable nucleated spleen ceils assayed on day indicated. 0 - - 0 , Normal,
SE-stimulated. © - - O , Normal, no SE added to cultures. & - - & , FV-infected, SE-stimulated.
A- -A, FV-infected, no SE added to cultures.
Fig. 2 shows the results of one of three similar experiments which followed the course of
the response in normal and infected cultures. Culture suspensions were prepared from the
pooled spleens of eight normal and eight infected mice. Four cultures for each category
(normal, infected, stimulated, unstimulated) were assayed each day for a week. During the
first 48 hr there were no significant differences in the rates of increase of PFC. After this
time while P F C counts in stimulated normal cultures continued to rise rapidly those in all
other categories rose more slowly and declined earlier.
Variation in primary responses
The experiments contributing to Table [ were performed over more than a year, during
which time may different batches of mice were used. In addition, while culture methods
remained unchanged, at least two batches of virus and of foetal calf serum were used, and
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 16:04:48
82
B.N. DRACOTT~ N. WEDDERBURN AND M. H. SALAMAN
the SE donor changed three times. Because such changes were from time to time inevitable,
it was important to determine whether the varying experimental results seen in Table i were
due to changes in reagents (serum, virus, antigen) or to differences in antigen responsiveness
between the various groups of mice used, and to determine the extent of individual variation
within such groups.
Mice for all experiments were weaned and sexed at 4 weeks of age and maintained in holding trays in batches of 30 or more. Mice from two such holding trays, arbitrarily called
group A and group B, were used for the experiment shown in Table 2. Normal mice from
group B, I I weeks older than group A, had twice given significantly greater responses than
the latter. However, preliminary experiments, which tested pairs of mice from every group
of mice then between 2 and 6 months old (including A and B), indicated that the size of the
PFC response is age- (and sex-) independent.
Table 2. Individual and group variation in primary responses to SE in vitro
of spleen cells from normal and 4-day FV-infected mice
PFC/Ios viable nucleated cells assayed after 5 days culture
<
Group A normal
Group B normal Group A infected Group B infected
__J<~_
A _ _
Mouse no.
+SE
-SE*
+SE
-S
I
313
552
75
89
II6
34
72
1-86
o"11
977
887
815
7o5
840
2"92
0"03
1o2
2
3
4
Geometric mean
Mean log PFC/Io6
+ Standard error
617
318
429
2"63
o'o8
49
55
44
59
1"77
o.o8
z.
+SE
-S
+SE
--SE
93
47
70
21
2i
2i
47
25
33
1"52
o'o9
59
36
42
1.62
o-1z
I5I
93
222
131
2.I2
0"09
9
2I
7
13
I.I I
o. 12
* No SE added to culture.
Eight mice were taken at random from each group: 4 animals from each were infected
with FV 4 days before culture, the remainder acting as normal controls. Stimulated and
unstimulated cultures were prepared from each spleen and assayed for PFC after 5 days
incubation. Table 2 shows that, in agreement with previous experiments, the stimulated
response of normal mice in group B was significantly higher than in group A. However, the
stimulated response of infected mice was less depressed in the latter group. Secondly, the
infected unstimulated response was significantly depressed in group B but not in group A.
Thirdly, individual variation was fairly small, being greatest in cultures from infected mice.
These results show that the differences in peak PFC numbers and in the degree of depression between experiments was due at least in part to variations between the groups of mice
used, and demonstrated the necessity that, as in the experiments forming Table I, mice for
any one experiment be taken from the same weaning group.
Responses of separated and recombined spleen fractions
Having established that the depression observed in vitro was essentially similar to that seen
in vivo, we next attempted to locate more precisely the site of the immune defect in infected
cultures. This was achieved by separating and variously recombining adherent macrophagelike cells (fraction M) and non-adherent lymphocyte-like cells (fraction L) from normal and
infected spleens. The results are shown in Table 3.
No fraction cultured alone produced a significant response compared with that of the
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 16:04:48
In vitro immune depression by Friend virus
83
unseparated suspension from which it was derived, although L fractions from normal primed
mice produced more PFC than L fractions from normal unprimed mice. In our hands the
secondary response in vitro of mice 4 months after exposure to a low primary dose of antigen
006 SE intravenously) was qualitatively similar to the primary response in vitro. Similar
findings have been made by other workers (Radcliffe & Axelrad, ~971 ; A. Munro, personal
communication).
Table 3. Response to S E in vitro of separated and recombined spleen cell
fractions from normal and 4-day FV-infected mice
PFC/Io ° viable cells assayed after 5 days culture
c
Cell status and treatment
Normal
Unseparated
M alone-~
L alone
M + normal L
Infected
Unseparated
M alonet
L alone
M +infected L
Expt I
Expt 2
Expt 3
Expt 4*
Expt 5*
726
2
56
I o66
247
Io
I4
843
i82
o
6
203
I8O3
5
319
1853
Io64
IO
I69
899
86
17
13
97
55
IO
15
91
76
lO
4
53
67
15
27
Iot
95
5
9
72
Normal M +infected L
95
14I
56
73
37
Infected M +normal L
850
699
267
3134
Io92
* Mice primed with one intravenous injection of io 6 SE 4 months previously.
? PFC per culture.
Combinations of uninfected M and L fractions responded as well as or better than the
suspensions from which they were derived. Combinations of infected M and L fractions, and
of normal M and infected L fractions, showed the same depressed response as an unseparated
infected suspension. However, infected fraction M plus normal fraction L responded as well
as or better than a normal suspension, either original or recombined. This clearly indicates
that the immunologically incapacitated cell type is located in the lymphocyte population
(fraction L) of an FV-infected spleen. It also shows that infection with FV in vivo does not
affect that part of the in vitro immune response which is mediated by macrophages.
DISCUSSION
The immune response of spleen cells from mice infected in vivo with FV, and stimulated
in vitro with SE, was found to be depressed when virus was injected 3 or more days before
the cells were taken for culture. The relationship between time of infection and degree of
depression was similar to that found in vivo, despite the fact that a number of influences
which may act on spleen cells in the intact animal must be absent from the simpler in vitro
environment. That a similar depression of the immune response is not always observed in
in vivo and in vitro systems was shown recently by Waterston (I97o), who found that prior
injection of pig erythrocytes depressed the in vivo PFC response to SE, while enhancing its
counterpart in vitro. This result is further evidence against the possibility that FV depresses
the immune response by acting as a competing antigen, which was discussed in a previous
paper (Wedderburn & Salaman, I968) and judged unlikely on our in vivo results.
The dilution of immunocompetent cells during the later stages of the disease was largely
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 16:04:48
84
B . N . D R A C O T T , N. W E D D E R B U R N
A N D M. H. S A L A M A N
avoided by working with mice which were injected with FV 4 days before their spleen cells
were taken for culture. However, it should be pointed out that, in vivo at least, if all the immune competent cells in a normal spleen are assumed to be still present, although diluted,
these cells are giving a severely depressed response throughout the disease. In this respect
FV differs from some other infective agents such as the Rowson Parr virus (Carter et al.
I97O), murine cytomegalovirus (Osborn, Blazkovec & Walker, 1968) and a murine plasmodium (Salaman, Wedderburn & Bruce-Chwatt, 1969) for all of which the period of severe
immune depression is brief.
The number of PFC per spleen produced in the primary immune response to SE in vivo
was shown not to be depressed in a FV-infected mouse until more that 48 hr had elapsed after
antigenic stimulation (Wedderburn & Salaman, 1968). A rather similar effect was observed
in the in vitro system: no significant differences were found between the responses of stimulated or unstimulated, infected or uninfected cultures for the first 48 hr. The question whether
the same mechanism underlies both these effects must await the results of further experiments, particularly those designed to elucidate the nature of the unstimulated response
in vitro.
Using the differential adherence cell-separation technique first described by Mosier (1967)
we have established that the immune defect in FV infection is located in the lymphocyte
population (non-adherent fraction)of the spleen. Biano et al. (197I), using the same technique, have shown that the immune defect in the spleens of tumour-bearing mice is also
located in this fraction.
Odaka & KOhler (1965) were able to induce persistent FV infection of macrophages in
vitro, and Odaka et al. 0966) suggested that FV infection might interfere with antigen
processing by macrophages. We have not determined whether macrophages from infected
spleens carry virus, but our results show that, infected or not, their immunological function
was not impaired. If these cells were infected, the immune competence of the normal lymphocytes with which they were combined was not altered by cross-infection.
The nature of the defect in the immunocompetent cells of spleens of FV infected mice is
not yet understood. The neoplastic cell or cells appear to belong to the erythroid series.
However, Hanna, Szakal & Tyndall (197o), who investigated the histological effect of the
very similar Rauscher virus, reported that the earliest proliferation of the virus occurred in
immunoblasts of the germinal centres. Chan et al. (I 968), who looked at the ultrastructure of
spleens of normal and FV-infected mice which had received SE, showed that while normal
spleens contained plasma cells and immature lymphocytes, in infected spleens there were
many immunoblasts containing virus particles together with a few plasma cells and lymphocytes in which no virus was seen.
There is evidence (Friedman & Ceglowski, 1968; Bennett & Steeves, 197o) that the function of a bone-marrow-derived spleen cell is affected by FV. The in vitro system described
above has been shown by several authors to require the co-operation of at least three different types of cells - one adherent cell and two different non-adherent lymphoid cells, one of
which is probably thymus, and the other bone marrow, derived. Experiments are now in
progress which are designed to determine whether the immune functions of either or both
of these types of splenic lymphocytes are affected by FV.
This work would not have been possible without the advice and encouragement of Professor
J. L. Turk and Drs A. Munro, Philippa Hunter, M. R. Salaman, E. M. Tucker and H.
Festenstein, to all of whom we extend our sincere thanks. We wish also to thank Mr C. Turton and Miss Diana Bullen of Wellcome Reagents Ltd for the provision of erythrocytes from
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 16:04:48
In vitro immune depression by Friend virus
85
individual sheep. Expert technical assistance was given by Mrs B. Adkins, Mrs J. Newton
and Mr A. Silcox.
The expenses of this research were partly met by a grant from the Cancer Research
Campaign. BND is supported by a grant from the Lillian May Coleman Fund for Cancer
Research.
REFERENCES
BENNETT, M. & STEEVES,R. A. (I970)- I m m u n o c o m p e t e n t cell functions in mice infected with Friend leukemia
virus. Journal of the National Cancer Institute 44, I IO7.
maNo, G., BROWN, R. L., JONES, E. E. & ROSFNOER, V. M. (I97i). ]The cell site o f the immunological defect in
tumor-bearing mice. Proceedings of the Society for Experimental Biology and Medicine 136 , 5o7.
CARTER, R. L., CHESTI~RMAN,F. C., ROWSON, K. E. K., SALAMAN, M. H. & WEDDERBURN, N. (I97O). A new virus
o f minimal pathogenicity associated with Friend virus. II. Histological changes and immunodepressive
effect. International Journal of Cancer 5, 103CEGLOWSKI,W. S. & FRIEDMAN,H. (1968). Immunosuppressive effects of Friend and Rauscher leukemia disease
viruses on cellular and humoral antibody formation. Journal of the National Cancer Institute 4 o, 983.
CHAN, G., RANCOURT, M. W., CEGLOWSKI, W. S. & FRIEDMAN, H. (I 968). Leukemia virus suppression o f antibody
forming cells: Ultrastructure o f infected spleens. Science, New York 159, 437.
DAWSON, e. J., FIELDSFEEL,A. H. & BOSTICK, W. L. (1963). Pathologic studies o f Friend virus leukemia in the
development o f a transplantable tumor in Balb/e mice. Cancer Research 23, 349DUTTON, R. W., MCCARTHY, M. M., M1SHELL,R. I. & RAIDT, D. J. (197o). Cell components in the immune response.
4. Relationships and possible interactions. Cellular Immunology x, I96.
FRIEDMAN, n. & CEGLOWSKI, W. S. 0968). Cellular basis for the immunosuppressive properties of a leukaemogenic virus. Nature, London 218, 1232.
FRIEND, C. (I957)- Cell-free transmission in adult mice o f a disease having the character o f a leukemia.
Journal of Experimental Medichze 1o5, 3o7.
HANNA, U. G., SZAKAL,A. K. & TYNDALL, R. L. (1970). Histoproliferative effect o f Rauscher leukemia virus on
lymphatic tissue: Histological a n d ultrastructural studies of germinal centers and their relation to
leukemogenesis. Cancer Research 3 o, I748.
nASKILL, J. S., BYRT, l'. & MARBROOK, J. 0970). In vitro and in vivo studies o f the immune response to sheep
erythrocytes using partically purified cell preparations. Journal of Experimental Medicine 131 , 57.
HIRANO, S., CEGLOWSKI, W. S., ALLEN, J. L. & FRIEDMAN, H. (I969). Effect of Friend leukemogenic virus on
antibody forming ceils to a bacterial somatic antigen. Journal of the National Cancer Institute 43, 1337JERNE, N. K. & NORDIN, A. A. (~963). Plaque formation in agar by single antibody-producing cells. Science,
New York 14o, 4o5.
MARBROOK, J. (I967). Primary immune response in cultures o f spleen cells. Lancet ii, I279.
METCALF, D., FURTH, J. & BUFFET, R. r. 0959). Pathogenesis of mouse leukemia caused by Friend virus. Cancer
Research 19, 52.
MILLIAN, S. J. & SCHAFrFER, M. (I968). Antibody production by mice infected with selected murine oncogenic
agents. Cancer zx, 989.
MIRAND, E. A. (I967). Virus-induced erythropoiesis in hypertransfused polycythemic mice. Science, New York
I 5 6 , 832,
MISHELL, R. I. & DUTTON, R. W. (1967). Immunization of dissociated spleen cell cultures from normal mice.
Journal of Experimental Medicine 126, 423.
MOSlER, O. E. (1967). A requirement for two cell types for antibody formation in vitro. Science, New York
x58, I573MOSIER, D. E. & COPPLFSON, L. W. (I968). A three-cell interaction required for the induction of the primary
immune response in vitro. Proceedings of the National Academy of Sciences of the United States of
America 6i, 542.
ODAKA, T., ISHII, H., YAMAURA, K. & YAMAMOTO, T. (I966). Inhibitory effect of Friend leukemia virus infection
on the antibody formation to sheep erythrocytes in mice. Japanese Journal of Experimental Medicine
36 , 277.
ODAKA, T. & K()HLER, K. 0965). ()ber Aufnahme und Vermehrung des Friend-Leukemic-Virus in Makrophagen. Zeitschrift fiir Naturforschung 2ob, 473OLD, L. J., CLARKE, D. A., BENACERRAF, B. & GOLDSMITH, M. (196o). The reticulo-endothelial system and the
neoplastic process. Annals of the New York Academy of Sciences 88, 264.
OSBORN, J. E., BLAZKOVEC, A. A. & WALKER, D. L. (1968). Immunosuppression during acute murine cytomegalovirus infection. Journal of Immunology ioo, 835.
RADCLIFFE, G. N. & AXELRAD,M. A. (197I). Immunological memory in vitro. Journal of Experimental Medicine
133, 846.
ROSSI, G. B., CUDKOWICZ, G. & FRIEND, C. (I970). Evidence for the transformation of spleen cells one day after
infection of mice with Friend leukemia virus. A u t o n o m o u s growth potential and expression of hybrid
resistance genes. Journal of Experimental Medicine 131, 765.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 16:04:48
86
B.N.
DRACOTT,
N. W E D D E R B U R N
AND
M. H. S A L A M A N
SALAMAN,M. H. & WEDD~RBURN, N. 0 966). T h e i m m u n o d e p r e s s i v e effect o f Friend virus. Immunology Io, 445.
SALAMAN, M. H., WEDDERBURN, N. & BRUCE-CHWATT, L. J. (I969). T h e i m m u n o d e p r e s s i v e effect o f a m u r i n e
p l a s m o d i u m a n d its interaction with m u r i n e oncogenic viruses. Journal of General Microbiology 59, 383.
TAN, T. & GORDON, J. (197 I). Participation of three cell types in the anti-sheep red blood cell response in vitro.
Journal of Experimental Medicine 133, 520.
WATERSTON, R. H. (I970). Antigen competition: a paradox. Science, New York x7o , I IO8.
WEDDERBURN, N. & SALAMAN,M. N. (2968). T h e i m m u n o - d e p r e s s i v e effect o f Friend virus. I[. R e d u c t i o n o f
splenic h a e m o l y s i n - p r o d u c i n g cells in p r i m a r y a n d secondary responses. Immunology xS, 439.
(Received 3o June I 9 7 t )
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 16:04:48