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(CANCER RESEARCH 39, 950-955, March 1979]
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Immunosuppressive Properties of a Virion Polypeptide, a 15,000-Dalton
Protein, from Feline Leukemia Virus1
Lawrence E. Mathes,2 Richard G. Olsen, Lynn C. Hebebrand, Edward A. Hoover, Joseph P. Schaller,
Patrick W. Adams, and W. S. Nichols
Department of Veterinary Pathobiology, College of Veterinary Medicine [L. E. M., A. G. 0., L. C. H., E. A. H., J. P. S., P. W. A.], Department of
Microbiology, College of Biological Sciences (L. E. M., A. G. 0., L. C. H., P. W. A.J, The Comprehensive Cancer Center (A. G. O.J, and Department of
Pathology, College of Medicine (W. S. NJ, The Ohio State University, Columbus, Ohio 43210
ABSTRACT
sarcoma induced by challenge with FeSV (19, 22). Recent
in vitro studies have shown that inactivatedmurine and
The 15,000-molecular-weight polypeptide (p15) of feline
leukemia virus (FeLV) was shown to impair normal lympho
cyte function in vitro and to abrogate immunity to feline
oncornavirus disease in vivo. FeLV p15 suppressed concan
avalin A-induced blast transformation of normal feline lym
phocytes by 68%, while other virion proteins had no effect.
p15 suppression was not due to toxicity, nor was p15 a
competitive inhibitor of concanavalin A binding. Capping of
receptors for concanavalin A on normal feline lymphocytes
also was inhibited by either inactivated FeLV or FeLV p15.
Groups of cats were immunized with either killed feline
oncomnavirus-associated cell membrane antigen bearing
tumor cells or tumor cells plus FeLV p15. After challenge
with feline sarcoma virus, three of four p15-treated cats
developed progressive fatal fibrosarcoma as compared to
one of five non-pl 5-treated cats. The cats receiving p15
also had lower cytotoxic antibody titers against feline on
comnavimus-associated cell membrane antigen (mean peak
titer, 1:6) than did the non-p15 group (1:74). These data
support the hypothesis that the immunosuppression in cats
infected with FeLV is mediated by FeLV p15.
INTRODUCTION
The immunosuppression associated with metrovirus infec
tions of several species (for review, see Ref. 5) generally
has been attributed
to impaired
lymphoid
function
second
ary to viral infection of lymphoid tissues. Several recent
reports,
however,
indicated
that impaired
immune function
can be induced by inactivated or attenuated retrovimuses in
the absence of infection. In one study, mice inoculated with
attenuated MuSV3 and later challenged with infectious
MuSV had a higher incidence of malignant disease than did
control mice (24). Likewise, immunization of cats with UV
inactivated FeLV caused abrogation of immunity to fibro
I Supported in part by USPHS Contract NO 1 5-3571 and CP-VO-81035-63
from the Division of Cancer Cause and Prevention of the Virus Cancer
Programand byGrantCA-15147-03
from the NationalCancerProgramof the
NIH.
2 To
whom
requests
for
reprints
should
be
addressed,
at
Department
of
Veterinary Pathobiology, The Ohio State University, 1925 Coffey Road,
Columbus, Ohio 43210.
3 The
abbreviations
used
are:
MuSV,
murine
sarcoma
virus;
FeLV,
feline
leukemia virus; FeSV, feline sarcoma virus; LBT, lymphocyte blast transfor
mation; SPF, specific-pathogen-free; Con A, concanavalin A; rH]dThd,
tritiated thymidine; p15, 15,000-dalton protein; TKE-D-Tx, 0.05 M Tns, 0.6 M
KCI, 0.01 N EDTA, 0.01 N dithiothreitol, and 1% Triton x-ioo buffer; p27,
27,000-dalton protein; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide
gel electrophoresis; FOCMA, feline oncornavirus-associated cell membrane
antigen ; FITC, fluorescein isothiocyanate.
Received June 21, 1978; accepted December 1. 1978.
950
feline leukemia viruses suppress at least one normal lym
phocyte function, that being phytomitogen-induced LBT (9,
12).
The present study was undertaken to investigate further
the lymphocyte unresponsiveness caused by exposure to
inactivated FeLV and to identify possible subvimal compo
nent(s) which may have a similar effect.
MATERIALSAND METHODS
Cats. All cats used in vaccination experiments and as
blood donors for the LBT assay were taken from The Ohio
State University, Department of Veterinary Pathobiology,
SPF cat colony (21).
LBT Assay. The LBT assaywas a modificationof that
described
by Cockerell
at al.
(2). Enriched
lymphocyte
preparations were obtained by centrifugation of heparin
ized blood through Ficoll-Hypaque gradients. One-tenth ml
of the cell suspensions (1 x 10@cells/mI) was mixed in
microtest plates (Falcon Plastics, Oxnard, Calif.) with 0.05
ml of an optimal concentration of Con A (Sigma Chemical
Co. , St. Louis, Mo.) (10 p.9/well) and 0.05 ml of the protein
being assayed for LBT inhibition. The plates were incubated
at 37°for 5 days. The protein preparations being assayed
for LBT inhibition previously had been dialyzed 3 times
against 400 volumes of minimal essential medium (suspen
sion) containing 1% antibiotics. In the Con A control wells,
the inhibitory protein was replaced with 0.05 ml complete
medium. In cell control wells, both Con A and inhibitory
proteins were replaced with complete medium. During the
final 18 hr of incubation, 0.1 ml of medium containing 0.5
p.Ci of [3H]dThd (6.7 Ci/mol;
New England Nuclear, Boston,
Mass.) was added . Cells were collected on glass filter paper
with a semiautomatic multiple processor (Otto Heller Co.,
Madison, Wis.) and assayed for radioactivity by liquid scm
tillation counter. Net cpm of quadmuplicate wells were
averaged to obtain mean cpm.
Virus. Productionand purificationof RickamdFeLV had
been described (17). The only addition to the procedure
was that purified virus was banded a second time on linear
sucrose gradient.
Virus Fractionation.The technicalaspectsof the proce
dure for purification of p15 from FeLV are described in
another report.4 In this method (see Chart 1), purified FeLV
(lOll particles/mi) was twice freeze-thawed and centrifuged
4 L.
E.
Mathes
and
R.
G.
Olsen.
Feline
leukemia
virus:
purification
and
characterization of polypeptide p15, manuscript in preparation.
CANCERRESEARCHVOL. 39
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Immunosuppression
I
-
78
23456
_
94,000
by FeLV p15
lymphoma. FL-74 cells were grown in suspension cultures
in 2-liter roller bottles as described previously (20). Cultures
were initiated at a concentration of 1.6 to 1.8 x 10@cells/mi
with 200 mI/bottle. Three days after transfer, an additional
200 ml of McCoy's
Medium
5A containing
15% fetal calf
were added. At 5 days, the cells were harvested by
. 67,000 serum
centrifugation; final cell counts were 7 to 13 x 10@cells/
S 43@00
@
@
@
@
p27@
30,000
20,100
p15
l4@400
bottle.
FL-74 cells were killed but not lysed by heating
in a 56°
water bath for 4 mm by the procedure of Heding et al. (13).
The FL-74 cell inoculum was prepared by emulsifying 5 x
108 heat-killed FL-74 cells (approximately 0.5 ml) with 0.5
ml 0.01 M Tris-HCI, pH 7.0-0.10 M NaCI-O.001 M EDTA buffer
and 1 ml complete Freund's adjuvant. The FL-74 cell plus
p15 inoculum was prepared by emulsifying 5 x 10@heat
killed FL-74 cells (0.5 ml) and either 300 or 100 p.g of p15
(0.5 ml) with 1 ml complete Freund's adjuvant. Cats were
given i.m. injections 3 times at biweekly intervals.
LymphocyteViability.Viabilityof lymphocyteswasdeter
mined by their capacity to exclude trypan blue dye.
Polyacrylamide Gel Analysis. PAGE was performed in
12-cm slab gels using 7.5% acrylamide with 0.375% bisac
Fig. 1. PAGE analysis of FeLV protein. Electrophoresis was in 7.5%
polyacrylamide gel in the presence of 0.1% SDS. 1, FeLV; 2, Fraction A; 3,
rylamide in a Tmis-acetate buffer containing 0.205 M Tris,
Fraction B; 4, Fraction C; 5, Fraction D; 6, purified p15; 7, purified p27; 8,
0.205
M acetic acid, and 0.1% SDS, pH 6.6. Protein samples
protein standards: phosphorylase b, 94,000; bovine serum albumin, 67,000;
were mixed with equal volumes of 0.2 M Tris, 0.02 M acetic
ovalbumin, 43,000; carbonic anhydrase, 30,000; soybean trypsin inhibitor,
20,100; cr-lactalbumin; 14,400.
acid, 0.1% SDS, and 0.1 M dithiothmeitol and heated to 100°
for 3 mm prior to electrophoresis. Gels were electropho
resed for 5 hr at 100 V.
at 100,000 x g for 90 mm with a SW27 rotor in a Beckman
Virus Inactivation. UV inactivation of FeLV was accom
L2 65-B ultracentrifuge (Beckman Instruments, Palo Alto,
plished with a surface dose rate of 150 ergs/sq mm/sec for
Calif.). The liquid was collected and stored at —90°
(Fraction
an accumulated total of 35,000 ergs/sq mm (25).
A). The protein pellets were resuspended and partially
51Cr Cytotoxicity Assay. 51Cr-labeled FL-74 cells were
solubilized in 2 ml of TKE-D-Tx buffer, pH 7.2, and incu
prepared by incubating 8 x 10@cells in 1 ml of McCoy's
bated at 37°for 1 hr. The soluble protein was separated
from the insoluble material by centrifugation at 100,000 x g Medium 5A (3-day-old cultures) with 0.2 ml of Na51CrO4
solution (approximately 200 p.Ci) at 37°for 1 hr with con
for 90 mm. The insoluble pellet was resuspended in TKE-D
Tx buffer and stored at —90°
(Fraction B). The liquid phase stant agitation. After 1 hr, the labeled cells were washed
was collected and extracted 8 to 10 times with 10 to 15 with 40 ml of cold McCoy's Medium 5A by centrifugation.
volumes of ether to remove the Triton X-100. By removal of The cell pellets were resuspended in 40 ml of cold media
and incubatedat 4°for 30 mm. Finally,
the cellswere
the Triton X-100, the p15 protein became insoluble. Follow
ing the final ether extraction, residual ether was removed centrifuged, and the pellets were resuspended in 1 ml of
by blowing a fine stream of nitrogen across the surface of media.
Serial 2-fold dilutions of feline serum (25 p.l) were made
the liquid. The insolubilized p15 was collected by centrifu
in U-bottomed microtiter plates (Cooke Engineering, Alex
gation at 100,000 x g for 90 mm. The liquid phase (Fraction
C) was removed for later use, and the pellet (Fraction D, andria, Va.) with 25-p.l Cooke microdiluters in McCoy's
Medium 5A containing 10% fetal calf serum as diluent.
p15 enriched) was redissolved in TKE-D-Tx buffer.
Purification of FeLV p15. p15 was purified from Fraction Added to the test serum were 50 p.1of rabbit serum as a
D by liquid column chromatography with a 2.5- x 90-cm source of complement (diluted 1:2) and 25 p.1containing 2
x 10@51Cm-labeledFL-74 cells. The plates were incubated at
Sephacryl 200 (Pharmacia, Upsala, Sweden) column equili
brated with modified TKE-D-Tx buffer (0.01 M Tris-HCI, 0.3 37°for 1 hr with periodic agitation. At the end of the
incubation period, the plates were centrifuged at 600 x g
M KCI, 0.01 M EDTA, 0.01 M dithiothreitol,
and 1% Triton X
for 5 mm in an International PR-6 centrifuge with No. 276
100), pH 7.2. A small amount of Fraction D was madiolabeled
head (International Equipment Co. , Needham Heights,
with 1251using the chloramine-T method (10) to facilitate
identification of protein peaks. A single major peak of Mass.) and carrier bucket (Cooke Engineering). Tissue
culture fluid containing released 51Cmwas collected using
highly purified p15 was resolved by this procedure.
the Titertek supemnatant collection system (Flow Laborato
Purification of FeLV p27. p27 was purified from Fraction
C by liquid column chromatography using a 2.5- x 90-cm ries, Rockville, Md.). The amount of 51Cr released was
scintillation counting with a Biogamma II
Sephadex G-150 column equilibrated with 0.01 M Tris-0.3 M determined by @‘
counter (Beckman Instruments, Palo Alto, Calif.). The cyto
KCI-0.01 M EDTA-0.01 M dithiothreitol, pH 7.2.
Preparationof FL-74 and FL-74 plus p15 Inocula. The toxic antibody titer was the reciprocal of the highest serum
dilution that produced 50% release of cell-bound 51Cm.
FL-74 cell line is feline lymphoblastoid cells, originally
Controls included target cells incubated with either corn
established by Theilen at a/. (23) from a FeLV-induced
MARCH 1979
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951
L.E. Mathes atal.
plement alone or test serum alone.
Indirect Membrane Immunofluorescence
rabbit anti-goat y-globulin (Miles Laboratories, Inc.). The
counterstain was 0.5% Evans blue in H2O.Cats were consid
FOCMA.The indirectmembraneimmunofluorescence
test ered to be FeLV vimemic if fluorescence was detected in any
for antibody to FOCMA was developed by Essex at a!. (8). of the blood leukocytes.
Live FL-74 cells grown in suspensions from 3- to 5-day-old
FITC-Con A Labeling of Lymphocytes for Capping. The
cultures were used as target cells. The cells were washed procedure used to assay capping of Con A receptors on
twice with Hanks' balanced salt solution and resuspended
feline lymphocytes was that of Dunlap at a/. (7). Lympho
at a concentration of 1 x 106 cell/mI. Serial 2-fold dilutions
cytes obtained by Ficoll-Hypaque gradient centrifugation of
of sera were made in microtiter plates using 50-p.l diluters.
peripheral blood were suspended at a concentration of 2 x
Fifty p.1of cell suspension were added to each well, and the 106 cells/mI
in minimum
essential
media
(Grand
Island
plateswere incubatedfor30 mm at 37°.
The plateswere Biological Co., Grand Island, N. Y.) containing FITC-Con A
then centrifuged, and the cell pellet was washed twice with
(Miles-Jeda, Israel), 50 p.g/ml. Various concentrations of
Hanks' balanced salt solution.
UV-inactivated FeLV or purified FeLV p15 were incubated
Fifty p.1 of FITC-conjugated rabbit antiserum to cat ‘y with the cells for 15 mm at 37°.Controls were incubated
globulin (Miles Laboratories, Inc. , Elkhart, Ind.) were then without virus or virus protein. Following incubation, the
added to each well and incubated as above. The cells then cells were washed twice with fresh minimum essential
were washed twice and examined for membrane fluomes media. Capping was determined by examining the cell
cence with a Zeiss Universal fluorescence microscope (Carl suspensions by UV microscopy. The percentage of cells
Zeiss Inc. , New York, N. Y.). The end-point titer was the last undergoing capping was calculated from counts of 100 to
dilution of serum for which membrane fluorescence could
200 cells.
be detected.
Test for
Test for FeLV Viremia. The test for FeLVviremiawas a
modification of the indirect immunofluorescence procedure
developed by Hardy et a/. (11). The presence of FeLV group
specific antigens in circulating leukocytes and/or platelets
correlates the presence of infectious virus in plasma. Blood
smears were fixed in absolute methanol. The primary me
agent was hyperimmune goat anti-FeLV serum which had
been repeatedly absorbed with normal cat blood until no
antibody reactivity could be detected with normal cat leu
kocytes (14). The secondary reagent was FITC-conjugated
RESULTS
Inhibition of LBT with Inactivated FeLV and Subviral
Componentsof FeLV. InactivatedFeLVand subvimal
frac
tions of FeLV were assayed for their ability to inhibit Con Astimulated LBT. Four subviral fractions of FeLV were de
mived, based on solubility, according to the fractionation
scheme shown in Chart 1 (see “Materials
and Methods―).
Crude fractions of viral protein were used in this initial
study so that minor components in the whole virus prepa
FeLV
(*1011PARTICLES/mI
PURIFIED
BY 2x SUCROSEDENSITY
GRADIENT
CENTRIFUGATION
FROZEN
THAWED
2x
ULTRACENTRI FUGATION
(00,000 G FOR I HOUR
SUPERNATANT1
FRACTION
RESUSPEND
(N
TKE-D-Tx
BUFFER
Chart 1. Scheme for separating FeLV into Fractions A
through D.
A]
INCUBATE AT
370 FOR I HOUR
ULTRACENTRI FUGATION
100,000 G FOR I HOUR
[PELLET
SUPER@IJATANT1
TFRACTIONB
ETHER EXTRACT
lOx
REMOVE ETHERBY
N2 BUBBLING
ULTRACENTRIFUGATION
100.000 FOR I HOUR
[PELLET
@RACTION
D
952
[SUPERNATAN
[FRACTIONC
CANCER RESEARCH VOL. 39
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Immunosupprassion
ration would not be lost in the process of ultrapurification
of specific proteins.
Lymphocytes of SPF cats were incubated with an opti
mum concentration of Con A and with either inactivated
FeLV or one of the 4 subviral fractions. The amount of
[3H]dThd incorporated into these cultures was compared to
that of cultures from the same cats incubated without the
indicated protein (Table 1). Inactivated FeLV reduced the
reduced the level of stimulation by a mean value of 68%.
p27 inhibited only 18%.
Lymphocyte Viability after Incubation with p15. To deter
mine if the reduction of [3H]dThd uptake in lymphocytes
treated with p15 was due to a cytotoxic effect of the protein,
lymphocytes from 2 cats were incubated under LBT condi
tions, but without Con A, for 5 days with 5 p.g of p15 per
well. In each case (Table 3), lymphocyte counts for tests
incorporation
incubated with p15 remained at approximately
number per well as did those of controls without
of [3H]dThd by an average of 43% (Table 1).
The Fraction D preparation also repressed LBT, in this
case, by an average of 41% (Table 1). Fractions A, B, and C,
however, did not significantly repress LBT (Table 1).
Polyacrylamide Gel Electrophoretic Analysis of Crude
FeLV Fractions. Protein samples from whole FeLV and
Fractions A, B, C, and D were analyzed by SDS-PAGE to
determine their protein composition. Fraction A was corn
posed of primarily higher-molecular-weight proteins (Fig.
1). Fraction B contained a mixture of proteins which were
insoluble under the extraction conditions in TKE-D-Tx
buffer. Fraction C contained p27 as its chief component in
addition to several minor component proteins. Fraction D
contained p15 as its major component. Ultrapurification of
p15 from Fraction D and p27 from Fraction C were accom
plished as described in ‘
‘Materials
and Methods.―
Inhibition of LBT by Purified p15 and p27. Highly purified
p15 or p27 were added to assays as previously done with
virus to determine
if either protein had inhibitory
properties
similar to whole FeLV or to FeLV Fraction D (Table 2).
Lymphocytes from 6 normal SPF cats were used for p15,
and those from 2 normal SPF cats were used for p27. p15
Repression
Table 1
of LBT by FeLV and FeLVprotein
of protein (100-p.l volume)and
an optimum dose
A (10 /Lg in 50 @l)were incubated
quadruplicate
with 1 x
10@lymphocytes/well
for 5 days in micrinotiter plates. [3HJdThd
wasadded
onday.Protein
the fourth
% inhibitiona
LBTWhole
(1 Mean
percentage
of
No.ofcatsMean
FeLVb
12.5cFraction
9.3FractionA
8.5FractionB
C
1.5FractionD
inhibition
of
A
LBT
cultures
with
7% capping
(50 to 80% reduction),
Table 2
of LBT with p15 and
3Effect
of cats used
lymphocyte do, nors
Table
cells!well
lymphocytes were seeded at a concentration
of 1 x 10@
toquadruplicate
in microtiter
plates. Five @gof p15 in media were added
anequivalent
wells. Controls, in quadruplicate wells, received
± S.D.
was determined
by
concentration/well
p151312
With p15
1.481409
1.58
(x
Without
1.36
1.31
Table 4
Effect of increased Con A concentration of p15-induced LBT
of repression
@gof p15 were added to each well of quadrupli
cate tests. Percentage of inhibition was determined by the formula:
.
.
.
.
/olnhlbltlOn
-
3H
-
cpm!welI
of
tests
incubated
with
p15
3Hcpm/well of tests Incubated without p15
Mean % inhibition
Con A concentration
LBT1099.02598.15096.2
(j.@g!weII)Mean
2
b Mean
lymphocytes
lymphocytesCat
of FeLV p15 on the viability of normal feline
50 j@g!welI). Five
7p27
percentage
whereas
Lymphocyte cultures from 4 cats were set up for LBT using
optimal (10 @g!weIl)and increased concentration of Con A (25 and
A(10
p15 and p27 (5 @g/welI)and an optimum dose of Con
7andj.@gin 50 s.d) were incubated with 1 x 10@lymphocytes from
wasadded
2 cats, respectively,
in quadruplicate
tests. [3H]dThd
on Day 4 of culture,
5.No. and cells were harvested on Day
a Mean
visible
inhibition
Repression
asPurified
ofpeptide polyLBTap15
of FeLV
A underwent
from 4 cats incubated with FITC-Con A plus p15 (50 p.g/rnI)
had 5 to 8% capping (50 to 83% reduction).
Incubation of lymphocytes with UV FeLV or p15 did not
interfere with FITC-Con A binding. The number of fluoresc
ing cells was not diminished as compared with controls
(data not shown).
Immunosuppressive Effects of p15 in Cats. This aspect
ofthestudywas undertakentodetermineifinoculating
p15
into cats that were simultaneously being immunized with
± S.D.
p27Purified
FITC-Con
incu
b UV-inactivated FeLV.
MARCH
treated
capping after a 15-mm incubation at 37°.Capping was
reduced when either UV FeLV or FeLV p15 was included in
the incubation mixture (Table 5). Lymphocytes from 6 cats
incubated with FITC-Con A plus FeLV (210 jig/mI) had 2 to
10@)Cat
bated with the indicated protein. Repression was determined
by
dividing the [3H]dThd uptake in the test culture by that of the
control cultures incubated without the indicated protein.
C Mean
lymphocytes
exclusion after 5 daysincubation.Lymphocyte
of
±
.4 ±
±
.5 ±
±13.3
Con
Test for Competitive Inhibition of Con A by p15. To
determine if p15 was a competitive inhibitor for Con A
binding, the amount of Con A per test was increased by a
factor of 2.5 and 5 (Table 4). The suppressive effect of p15
was not altered by higher concentrations of Con A.
Effect of Inactivated FeLV and p15 on Capping of FITC
Con A-treated Lymphocytes. Ten to 33% of normal cat
trypanblue volume of media. Cell viability
343.1
21
23.5
21
241.1
of
the same
p15.
fractions
Dilutions
ofCon
@
by FeLV p15
% inhibition
of
18.0±2.8
of Con
A-LBT
cultures.
1979
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1979 American Association for Cancer Research.
953
L.E. Mathas etal.
killed tumor cells (FL-74 cells) would alter the immune
response of the cats to the tumor cells. The criteria used to
evaluate the tumor cell immune response were (a) cytotoxic
antibody response to FOCMA and (b) resistance to FeSV
carcinogenesis.
Kittens from each of 2 litters were apportioned equally
into either of 2 treatment
groups
receiving
The tumor incidence in the cats given p15 plus FL-74
cells (3 of 4) was greater than that in cats vaccinated with
FL-74 cells alone (1 of 5) (Table 6). The mean peak cytotoxic
antibody titers before and after challenge differed signifi
cantly between the 2 groups. The highest mean titer for the
cats receiving p15 plus FL-74 cells was 1 :6 before challenge
and 1:3 after challenge as compared with 1:74 and 1:18,
(a) heat-killed
FL-74 cells or (b) heat-killed FL-74 cells plus FeLV p15. All
respectively,
kittens
2 shows the mean cytotoxic antibody titers of the 2 groups
of cats over the 8-week period prior to challenge. The
cytotoxic antibody titers were significantly
different
received
3 inoculations
on a biweekly
schedule
beginning at 4 weeks of age. At 12 weeks of age, all cats
were challenged
with FeSV at a dose previously
shown to
produce a 75% incidence of progressive fibrosarcoma
age-matched cats.
in
Table 5
Effect of UV FeLVand FeLVp15 on the capping reaction of Con A
receptors
lymphocytes%
on cat
cappingUV-inactivated
of lymphocytesundergoing
FeLV
at following protein
concentration
(/Lg/
ml)
FeLV-p15 at follow
FL-74 cells alone. Chart
throughout
this period. The incidence
groups was not significantly
different
of virernia in the 2
(4 of 4 for the p15
plusFL-74cellgroup and 3 of 5 forthe catsgivenFL-74
cells alone). High cytotoxic antibody titers in cats given FL
74 cells alone appeared to correlate with capacity to clear
FeLV virernia (Table 6).
DISCUSSION
Ifl@ protein concen
tration (j.@g!mI)
In this study,
inactivated
whole FeLV and subviral
corn
ponents of FeLV were analyzed for their inhibitory effect on
the Con A LBT response using lymphocytes from normal
SPF cats. Interference with lymphocytic function as deter
mined in the LBT assay was found with whole virus and
Con
507771
Cattrola
for the cats receiving
21
117
210
12.5
25
10
13
10
2
2
NT
2
2
3
NTb
NT
NT
NT
NT
NT
NT97417
14
5
4
NT
NT
NT902B15
NT43033
16
14
4
5
5
7
NT
NT
NT
NT
with other protein fractions of FeLV including purified p27.
The inhibition appeared not to be due to p15 toxicity or to
620224
NT
NT
NT
14
10
appeared to contain p15 by PAGE analysis (Fig. 1), its
568523
NT
NT
NT
13
10
NT72216
NT
NT
NT
8
9
NT
NT
14
10
insoluble form, due to association
or aggregation
with
other proteins, apparently prevented the p15 from having
biological effects. Alternately, the p15 protein appearing in
Fraction B may be a different protein than that in Fraction
NT1014B17
NT1013B20
NT792B21
0
a Percentage
of
cells
undergoing
NT
capping
for control
with Fraction D containing
competition
8
cultures
from each cat incubated with FITC-Con A but without inhibitory
pri@tein.
the p15 protein of FeLV, but not
for Con A binding
Fraction
B
D, but having the same migrating pattern in SDS-PAGE.
The fact that 2 p15 proteins
NT, not tested.
sites. Although
(pl5E
and pl5C)
have been
Table 6
Immunobiological response
of cats inoculatedwith
p15FL-74
FL-74 celi alone or FL-74 cells plus
cells + p15FL-74
aloneHighestHighestcytotoxiccytotoxicantibody
cells
titerTumorFeLVTumorFeLVCatBCatiterantibody
developmentviremia618-18
AC
P+618-28
R+618-364
R+618-464
R+!—618-564
R+!—678-12
mean cyto
AC
8
128
128
P+678-28
P+678-364
P+678-4128
R+Geometric
2.2toxic
developmentviremiaBC
8
8
2
2
4
4
±1 .2 18 ±
6 ±1 .7 3 ±1 .874
titer%progressivetumor7520%
antibody
0060
viremic cats1
a BC,
954
before
FeSV
challenge;
AC,
after
FeSV
challenge;
P, progressive
tumor;
R, regressed
tumor.
CANCER
RESEARCH
VOL. 39
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1979 American Association for Cancer Research.
Immunosupprassion
by FeLV p15
REFERENCES
cg
0
-J
w
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5
I-
0
0
z
‘C
4
1976.
3
U
0
0
2
I-
U
@
4
5
6
7
8
9
II
2
Gruneand Stratton,1975.
AGE OF CAT (WEEKS)
Chart 2. Cytotoxic antibody response of cats inoculated with 100 to 300
I@0 of FeLV
p15 plus
5 x 10. heat-killed
FL-74
cells
(0)
or 5 x 10. heat-killed
FL-74 cell alone (•).Cats were inoculated at 4, 6, and 8 weeks of age.
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described for murine leukemia virus (15, 16) lends credence
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FeLV and FeLV p15 caused alterations of cell membrane
functions as indicated by interference of FeLV p15 with the
normal capping process.
The artificial nature of the in vitro test for lymphocyte
functions makes it difficult to compare inhibition of LBT
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fections. Therefore, in orderto determine if p15 had biolog
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same time they were being vaccinated with heat-killed FL
74 cells. Vaccinating cats with FL-74 cells had previously
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toxic antibody response to FOCMA was reduced signifi
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significantly increased after FeSV challenge.
The combined results of the in vitro and in vivo studies
indicate that FeLV p15 causes alterations in lymphocyte
function which ultimately can affect the magnitude of the
immune response. This immunosuppressive property of
p15 would be beneficial to FeLV in terms of survival in
nature and pathogenesis of infection and disease. p15
expression at the initial site of viral replication could sup
press local immunological functions, allowing FeLV-in
fected cells to escape surveillance. With the development
of FeLV viremia, imrnunosuppression would become sys
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the growth
and survival
of neoplastic
clones of cells. Other investigators have detected immuno
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possibly endogenous) metmovirusescarry a structural corn
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local immune function at the site of turnorigenesis and
eventually may mediate overall irnrnunosuppression.
ACKNOWLEDGMENTS
We gratefully acknowledge Ken Milliser, Jo Ellen Dunlap, and Lilly
Romvary for their excellent technical assistance.
MARCH
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1979 American Association for Cancer Research.
955
Immunosuppressive Properties of a Virion Polypeptide, a
15,000-Dalton Protein, from Feline Leukemia Virus
Lawrence E. Mathes, Richard G. Olsen, Lynn C. Hebebrand, et al.
Cancer Res 1979;39:950-955.
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