Download Evaluation of the Cell-mediated Immune

[CANCER
RESEARCH
35, 2600-2608,
September
1975]
Evaluation of the Cell-mediated Immune Response to Murine
Sarcoma Virus by [‘251]Iododeoxyuridine
Assay and Comparison
with Chromium 51 and Microcytotoxicity Assays
Giuseppe Fossati,' Howard T. Holden,2 and Ronald B. Herberman3
Laboratory
of Immunodiagnosis,
National Cancer Institute, Bethesda, Maryland 200/4
the disease. Recently, detailed analyses of the primary cell
mediated immune response to MSV have been performed
The cell-mediated immune response of CS7BL/6 mice to by different laboratories (13, 14, 22, 23, 26). Nevertheless,
murine sarcoma virus (MSV) was examined by the [125I]@ the results obtained were not concordant. With CRA, the
iododeoxyuridine
release cytotoxicity assay using MSV
cell-mediated response appeared at 7 to 8 days after virus
induced sarcoma tissue culture cell lines as target cells. inoculation, reached a peak at 13 to 14 days, and then
Cellular cytotoxicity was detected as early as 3 days after declined rapidly (23, 26). In contrast, with visual MCA the
virus inoculation. Most mice assayed between 12 and 17 cytotoxic activity of lymphoid cells was biphasic, showing
days after MSV inoculation gave positive results with peaks in early and late periods after virus inoculation and
maximum levels of activity present on Days 13 and 14. negative results at 13 to 14 days (22, 33). Because of these
apparently discordant results, it was of interest to compare
Reactivity was frequently detected for up to 100 days after
MSV inoculation, although at low levels (5 to 10%). directly different assays for cellular immunity. A previous
Additional
experiments
comparing
the kinetics of the comparative
study of the cellular immunity to a Gross
virus-induced lymphoma in rats showed some correlation
cellular response as measured by different in vitro cytotox
icity assays were performed. The results showed a good between CRA and MCA, and many of the differences could
direct correlation between the [‘25ljiododeoxyuridine re
be accounted for by the different incubation periods in the
lease assay and a 51Cr release assay. A similar pattern of assays (40).
reactivity was also observed when the cellular response was
In this study, in addition to CRA and MCA, the
measured by a visual microcytotoxicity
assay, although
emphasis was placed on an assay in which cytotoxicity was
reactivity dropped off more rapidly and became undetecta
measured by the release of [‘251]IUdR. This assay has
ble in most instances by 20 days after injection of MSV.
previously been useful in detecting cell-mediated immunity
Studies on effector cell type revealed that cytotoxicity in all in both experimental and human tumor systems (4, 30, 38).
three assays was T-cell dependent, being eliminated by Like the MCA, it has a long incubation period and has been
treatment with anti-O plus complement. Macrophages did thought to be a more objective and quantitative equivalent
not appear to play a role, since treatment with carbonyl iron of MCA.
and magnet had no effect.
SUMMARY
MATERIALS
INTRODUCTION
Injection of MSV4.into adult mice induces tumors at the
site of inoculation, which undergo subsequent regression in
most animals (1 1). By in vivo experiments, this regression
has been shown to be mediated by immunological reactions
(2, 3, 6—11, 24, 25, 32, 36, 37). These studies, however, ex
amined only a few selected periods in the natural course of
1 Present
Tumori,
address:
Instituto
20133 Milano,
2 Supported
by
NIH
Nazionale
per
to
Studio
e
Ia
Cura
dci
Italy.
Fellowship
CA-5536l
from
the
National
Cancer
Institute.
S To
4 The
whom
requests
abbreviations
for
reprints
used
are:
should
MSV,
be
sent.
murine
sarcoma
virus;
CRA,
51Cr
release cytotoxicity assay; MCA, microcytotoxicity
assay; [‘261]IUdR,
[125I]iododeoxyuridmne;
FBS, fetal bovine serum; PBS, 0.007 M phos
phate-buffered solution of0.85% NaCI, pH 7.4.
Received October
2600
9, 1974; accepted June 9, 1975.
AND METHODS
Animals. Male CS7BL/6N (hereafter called B6) mice, 3
to S months old, were obtained from the Rodent and Rabbit
Production Section, Division of Research Services, NIH,
and from the Mammalian Genetics and Animal Production
Section of the Division of Cancer Treatment,
National
Cancer Institute, Bethesda, Md.
Viruses. LMV-4 and DMV-6, 2 stocks of the Moloney
strain of MSV (23), prepared from that originally obtained
by Dr. A. Fefer from Dr. Moloney's
laboratory
(SVRP-l 14) were used.
Tumor Cells. MSB and MSC tumor cells were estab
lished tissue culture lines derived from MSV sarcomas
induced in B6 and BALB/c mice, respectively. They were
kindly provided by Dr. Gary Pearson, National Cancer
Institute. Both of these cell lines release MSV and are
tumorigemc in vivo (28, 32).
CANCER RESEARCH
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
VOL. 35
Ce//-mediated
@
Immune
Response
to MS V
thistocytoma
were washed an additional time with PBS, combining
P815 tumor cells were a transplantable
DBA/2 mas
theused
with the cell pellet (“cells―).
Samples containing
and were grown in ascites form (5). The cells werefluid
placedafter
supernatant as well as those with cells were
both immediately after removal from the animals andoriginal
wasRBL-5
a well-type y scintillation counter, and each sample
several passages in tissue culture.in
(cpm).able cells were a Rauscher virus-induced transplant
counted for 1 mm
ofform,
P815 cells, 10 cells were incubated in 1 ml
lymphoma in B6 mice (29) and were grown in ascitesFor
wereInoculations.
with 1.0 @sCiof [‘26IJIUdRfor 6 hr. Then they
as well as in tissue culture.medium
athe
Animals were inoculated i.m. with MSV inwashed 3 times, counted, and resuspended in medium at
of l0@cells/mI. One-tenth ml of this suspen
thigh. Detectable tumors at the site of inoculation wereconcentration
attackerreached
was seeded in each well and 0.5 to 1.0 x l0@
seen approximately
7 to 8 days after virus administration,sion
wereregressed
were added in 0.4 ml of medium. The plates
their maximum size by 13 to 14 days, and thencells
rockingage-matched
at 37°in 5% CO2 for 16 to 20 hr on a
by 18 to 20 days. Control animals were normal,incubated
butgroup
(the P815 cells did not become adherent
mice receiving no inoculation. In addition, oneplatform
fromcells,
in suspension). The cells and supernatant
received a single, i.p. injection of 3 x 10@P8 15 ascitesremained
and the animals were tested 22 and 27 days later.such
cells were harvested as above, except that no trypsini
samplesthe
Lymphocytes. Axillary, inguinal, and mesenteric (and inzation
was needed for these suspension cells. The
ofmice
1st set of experiments also cervical) lymph nodes fromwith
P815 ascites cells were counted for 10 mm because
in experimental
and control groups were removedtheir
[‘26I]IUdR.under
low incorporation of
as:ing sterile conditions and were placed in medium contain
The percentage of cytotoxicity was calculated
(NIHMedia
Roswell Park Memorial Institute Medium 1640
% cytotoxicity
IslandBiological
Unit) with 20% heat-inactivated
FBS (Grand
(100IU/ml), Co., Grand Island, N. Y.), penicillin
cpm in cells
and streptomycin (100 @g/m1).The lymph nodes
=100
x 100
were teased apart, and the cell suspension and
fragmentswere
cpm in cells + cpm in supernatant
Aftercentrifugation,
passed through a double cotton gauze sponge.
the cells were resuspended in fresh mediumAll
meansand
tests were performed in quadruplicate,
and the
counted. Pooled lymphocytes of at least 2 immune miceand
inwere
standard errors of the percentage of cytotoxicity
used in each test, and pooled lymphocytes from at leastthe
calculated.2
experimental and the control groups were
age-matched,
syngeneic mice were used as controls.Experimental
results were expressed as percentage of cel
%manner
cytotoxicity
± S.E. with the normal control
Unless otherwise stated, spleen cells prepared in a similarlular
subtracted
out. Results were analyzed for
were used in the CRA.cytotoxicity
allAssays
significance by Student's I test. In these experiments,
cytotoxicity levels of 4% or more were significant
for Cell-mediated Cytotoxicitycellular
were[‘25I]IUdR
at a level ofp < 0.05 and some values between 2 to 4%
significant. In each test 4 replicates of target cells
Release Assay. This test was done in Linbro
with medium only were also included. The normal control
FB-l6-24-TC
tissue culture plates as previously described
cytotoxicity
in the experiments
performed
with MSB
(27). From 10,000 to 20,000 MSC or MSB target cells in 1 cells averaged 16.0%, with a range of2.0 to 35.2%.
ml of medium were seeded in each well, and the plates were
CRA. This test was performed as previously described
then incubated overnight at 37° in a moist 5% CO2
(23, 3 1). Briefly, 1 x l0@ spleen cells in Roswell Park
atmosphere. The medium was then removed by aspiration,also Memorial Institute Medium 1640 with 20% FBS
wereand
1 ml of fresh medium containing 0.5 ,tzCi of [‘251]IUdR
with 5 x l0@ 5'Cr-labeled RBL-5 target cells
(Amersham/Searle,
Arlington Heights, Ill.) were added to
(attacker:target
cell ratio of 200:1) for 4 hr at 37°in a
each well for 6 hr. The medium was then aspirated and
CO2 atmosphere
on a rocking platform. All tests
excess radioactivity was removed by washing 3 times with
were performed
in quadruplicate.
The amount of cell
Hanks' balanced salt solution plus 10% FBS. Immediately
lysis was determined by the cpm of 51Cr released into the
after the last wash, lymphocytes were added to labeled
calculatedthe
supernatant
fluid. The percentage of lysis was
target cells at a 500 to 1000: 1 ratio in 1.5 ml of medium, andincubated
as:
lymphocyte:-target
plates were incubated for 40 to 48 hr. The
cell ratio was estimated according to the number of
Lysis = cpm 51Cr released in experimental
group < 100
target cells, which became adherent after plating, calculated
cpm 51Cr released from cells by 4x freeze
initially for each cell line in the 1st experiments. After.
well,thawand
incubation the supernatant was aspirated from each
Thisfluid
the attached cells were washed once with PBS.
Controls for background release of 5'Cr for each experi
cellswere
was centrifuged and the supernatant and pelleted
ment included incubation
of labeled target cells with
saved.Then
PBSsolution
1.5 ml of EDTA-trypsin
solution (Dulbecco
autologous,
unlabeled target cells. Experimental
results
were expressed as corrected percentage of cytotoxicity by
EDTA)were
containing 0.25% crude trypsin and 0.01%
cellsuspension
added to each well to detach the cells. The
the subtraction of the background 5'Cr release. Differences
between experimental and control groups were analyzed for
was added to the 1st cell pellet, and the wells
SEPTEMBER
1975
2601
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
G. Fossati et a!.
significance with Student's I test. A test was considered
positive ifp < 0.05.
MCA. The method used was basically that described by
Hellström et a!. (15). Approximately 50 target cells (MSB)
were seeded into each well of 96-well plastic Falcon
Microtest II (No. 3040) culture plates. After overnight
incubation, medium was discarded and lymphocytes were
added in varying numbers. With this assay the attacker:tar
get cell ratio was calculated on the basis of the number of
target cells initially seeded. Eight replicates were used for
each lymphocyte preparation. The plates were incubated at
370
in
a
5%
CO3
atmosphere
for
40
to
48
hr.
The
wells
were
then washed carefully with PBS, and the cells remaining
adherent to the plates were stained with Giemsa and
counted under a microscope. Percentage of cytotoxicity was
computed by the following formula:
% cytotoxicity =
NL —IL
NL
x 100
where NL is the mean of surviving target cells after
incubation with normal lymphocytes and IL is the mean of
surviving target cells after incubation with immune lympho
cytes. Significance was evaluated by Student's t test.
Treatmentof EffectorCells with Anti-OAntibody.AKR
anti-U C3H antibody was produced by a procedure similar
to that of Reif and Allen (35) that has been described in
detail elsewhere ( 17). The main specificity of this reagent
has been previously demonstrated (17). For further demon
stration of the selective functional inactivation of T-cells
with this reagent, B6 lymph node cells were treated with
anti-O antibody plus complement and tested for proliferative
response to mitogens. This treatment almost completely
eliminated
reactivity
to phytohemagglutinin
and con
canavalin A (2 T-cell mitogens) while it did not affect
stimulation by endotoxin, a B-cell mitogen (H. Kirchner
and H. T. Holden, unpublished results).
For determination of the effect of anti-O antibody on the
cytotoxic response, lymph node cells were incubated for 30
mm at room temperature
with a final dilution of 1:2 of
antibody. The cells were washed once, resuspended in a 1:4
dilution of fresh rabbit serum as a source of complement,
and then incubated at 37° for 45 mm. Controls were
incubated at room temperature for 30 mm and then with
rabbit serum at 37°for 45 mm. After 2 additional washings,
the cells were counted, adjusted to the appropriate viable
cell concentrations, and tested for residual cytotoxic activity
in all 3 assays.
Removal of Phagocytic Cells by the Iron-Magnet Tech
nique.Phagocyticcells wereremovedfromthe lymphnode
virtually eliminates suppressor
activity (21) by macrophages.
(20) and growth-inhibitory
RESULTS
Results of [‘25I}IUdR
Release Assay after MSV Injec
tion.
In order
to determine
whether
[‘25I]IUdR release
assay was useful in detecting cell-mediated immunity in
the mouse MSV system, we performed preliminary ex
periments testing the cytotoxicity
of lymphocytes from
some MSV-infected animals, at different times after virus
inoculation, against MSB and MSC target cells. As shown
in Chart 1, cellular activity against MSB cells was de
tected on Day 9, rose to a peak on Day 14, and then de
dined on Day 16 and became negative by Day 24. The
curve obtained with MSC cells had the same shape but
lower cytotoxicity
levels, indicating that this allogeneic
cell line was less susceptible to lysis by immune lympho
cytes from B6 animals. Subsequent
experiments
were
performed with MSB cells, with a lymphocyte:target
cell
ratio ofSOO: I and with an incubation period of42 hr.
Experiments
were then performed
to determine
the
influence of the time of incubation on the cellular cytotoxic
ity results. When assays were terminated after 20 and 42 hr
of incubation (Table I), considerably different results were
seen. Cytotoxic activity was detectable at 20 hr but at low
levels. The amount of cytotoxicity
increased when the
incubation time was extended to 42 hr. The level of detected
activity was also affected by the lymphocyte:target
cell
ratios. As shown in Chart 2, percentage of cytotoxicity
increased with the amount of lymphocytes added, both in
the control and experimental groups. However, the percent
age of experimental cytotoxicity above the normal control
did not vary much, as the attacker:target
cell ratio increased
from 500 to 1500: 1. Subsequent experiments
were per
formed with a cell ratio of 500 to 1000: 1 and an incubation
period of 40 to 48 hr.
w
C/,
I1
CU
0
C0
C>.
U
4
-4
preparations
as previously described (20). Briefly, lymph
w
node cells were mixed with carbonyl iron (Technicon
Instruments Corp., Tarrytown, N. Y.) and incubated at 37°
for 30 mm. The tube containing the cell suspension was
placed over a magnet and the cells in the supernatant, which
5
20
25
30
35
40
45
did not ingest the iron particles, were harvested with a
DAYS
AFTER
VIRUS
INOCULATION
Pasteur pipet. Cells were washed and then used in the
Chart 1. Kinetics ofcellular cytotoxicity in MSV-infected mice against
assays. The carbonyl iron-treated cell suspension contained
MSB (0) and MSC (•)target cells in the [‘°51]IUdR
release assay.
less than 1% macrophages (12). Furthermore, this technique
Attacker:target
cell ratio, 500: I.
-4
-4
U
2602
CANCER RESEARCH
VOL. 35
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@
I
@
: @.-
I@
Ce//-mediated
Table I
Etiect of incubation on cvtotoxic activity of l@'mphoci'tes from
MS V-infected mice against MSB target cells
Lymph node cells from mice inoculated with MSV were tested for their
cytotoxicity against MSB cells in [‘°‘l]lUdR
release assay with the dif
ferent incubation periods.
cellularcytotoxicity20
Days after virus
incubation153.4
inoculation%
0.8aIS6.1
[email protected]
a Mean ±SE.
(p < 0.05).
hr incubation42
hr
±0.5°5.5
±[email protected]±
±0.2°9.2±0.2a
Experimental
value significantly
±
above normal control
-
03 40
x
0
-
30
-
20
-
f--.
4
-4
-4
U
0 -
0—
0
250
500
000
LYMPHOCYTETARGET CELL RATtO
On 2 occasions cytotoxicity
4 days
of Cytotoxicity
after
virus
inoculation.
in [‘25I]IUdR Assay.
Specificity studies were performed with P8 15 mastocytoma
cells, since these cells have been shown to be good target
cells for cellular cytotoxicity studies (I). Lymphocytes from
MSV-infected mice and from B6 mice given injections of
allogeneic P815 ascites cells were tested against both MSB
and P81 5 ascites cells. P81 5-immune animals showed good
activity against P8 15 ascites target cells, whereas no
reactivity was detected against MSB cells (Table 2). Lymph
node cells from MSV-immune mice, which were cytotoxic
for MSB cells, had no killing effect against P815 ascites
cells.
@T@@oonegative results were obtained in [‘25I]IUdR
releaseassay.
Chart 2. Cytotoxic activity of lymphocytes from MSV-infected mice,
14 days after virus inoculation (S), and from normal mice (0) against
MSB cells in the [1251]IUdR release assay.
@
at 3 and
in the [‘25I[IUdR release assay were also assayed at the
same time in CRA and in MCA. Table 3 shows the results
of the cellular cytotoxicity of MSV-infected mice and of
mice inoculated with P815 ascites cells, measured by
[‘25I]IUdR release assay and CRA against different target
cells. In general, good correlation between the results of the
2 assays was observed. Qualitative concordance (statisti
cally significant results in both tests) was found in 9 of I 3
tests performed. At high cytotoxic activity, there was also
good quantitative concordance, except in 2 cases. However,
when the level of cytotoxicity was low in CRA, lower or
C0
C-
@
Specificity
cytotoxicity.
early,
Comparison of [‘25I]IUdRRelease Assay with Other
Cytotoxicity Assays. Some of the mice tested for reactivity
C-
U
detected
to MS V
Further experiments were performed with P8 15 target
60 —
50
was
Response
cells after several passages in tissue culture. A cross-reactiv
ity was then observed. In fact, lymphocytes from MSV
immune animals showed a higher percentage of cytotoxicity
against P81 5 tissue culture cells than did lymphocytes from
animals immunized against P815 ascites cells. Low, but
significant, activity was detected by P815-immune mice on
MSB cells.
70 —
@
had no significant
Immune
The results of all these experiments are shown in Chart 3.
Lymphocyte cytotoxicity of MSV-infected mice was tested
at 28 different time points after virus inoculation starting on
Day 3 and continuing over a period of 100 days. There was a
considerable
variability in cellular cytotoxicity
ranging
between —16.0 and 49.6%. For some of the time points
after virus inoculation, repeated assays for cell-mediated
cytotoxicity
were performed.
Here again there was a
noticeable variability from one experiment to the next.
At 14 days after MSV inoculation, 15 different experi
ments were performed and the percentage of cytotoxicity
observed varied between 3.7 and 49.6. In contrast, closer
results were usually obtained on days where there was less
cytotoxicity.
Despite this variability in the results, there was a constant
significant cytotoxicity of all animals tested between 12 and
17 days. After Day 21, until Day 100, low levels of
cytotoxicity were often observed, although many animals
SEPTEMBER
1975
Low-level reactivity persisted for over 8 weeks regardless of
the assay used.
Although the 2 assays as performed appeared to give
50T
4O@S
>0
.
020—
0
..
.,O.—
•
-2O@@
0
S
-@—@__i@
5
‘0
5
20
DAYS
25
AFTER
30
ViRUS
I
35
40
70
00
iNOCULATiON
Chart 3. Cytotoxic activity of lymphocytes from MSV-infected mice
tested against MSB target cells in the [‘26I]IUdRrelease assay at different
days after virus inoculation.
2603
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
G.Fossatieta!.
Table 2
Specificity of cell-mediated cytotoxicitv in [‘251]IUdRrelease assay
Lymph node cells from mice immunized with either MSV or P815 ascites cells were tested for
their cytotoxic activity against MSB. P815 tissue culture, or P815 ascites cells.
cytotoxicityP815mice
after%
Lymphocytes fromDays
(ascites)MSV124.6
inoculated withinoculationMSB(tissue
culture)P815
±2.1MSV1528.6
1.1P8l5(ascites)27—5.4
[email protected]
0.8bP815(ascites)225.9
a Mean
±[email protected]
±1.6'—0.3
±2.116.0
±[email protected]
±1.0―18.2±
±
±
±
l.4b
± SE.
b Significant
cytotoxicity
above
the
control
(p
<
0.05).
Table 3
Comparison of the [‘251]JUdRrelease assay with the CRA: cvtotoxicitv against different target cells
B6 animals were immunized with MSV i.m. or P815 i.p. At different intervals after inoculation, lymphocytes were harvested and tested for cytotoxic
activity against RBL-5, P815 tissue culture, or P815 ascites.
cytotoxicity[‘25I]IUdR
(ascites)[‘25l]IUdR
I 5 (tissue culture)P8
Lymphocytesfrom
inoculation%
miceCRAMSV
inoculated withDaysafter
CRAP8
release assayRBL-5
±l.l―@'
MSV
27
1.3 ±0.7
@Ø4b
32.2 ±0.51
20.0±o5b
21.3±0.7b
11.7±0.2@@
16.3±0.2@@
7.4 ±0.2b
31.5±0.4@'
3.5±0.2'
25.5 ±0.5'
MSV
48
5.7 ±0.6―
11.5
P815(ascites)
P8l5(ascites)4
22
2713.1
4.6 ±2.5'
MSV
12
MSV
MSV
14
14
36.9 ±5.4@
20.4 ±53I@
MSV
14
13.2±I.4b
MSV
14
3.7 ±1.6
MSV
MSV
16
21
34.0 ±0.9―
—5.8±1.9
a Mean
release assayCRA[‘25lJIUdR
release assay
±2.1
±0.8@
±0.3'
2.6±0.3'
@Ø3I@
3.3±0.2b
18.2±1.4'22.3 19.7±0.2'1.4
16.0±1.2'
4.4 ±0.l@'26.3
5.9 ±1.0'
—5.4
±2.111.7
2.8±0.5k
41.2±0.3'
62.1±1.0'
± SE.
b Experimental
value
significantly
C Lymph
from
2 animals
nodes
above
pooled
the
for
control
[‘25I]IUdR
(p
<
0.05).
release
assay
but
correlative results, the [‘25I]IUdR release assay was per
formed with lymph node cells, while the CRA used spleen
cells as the effector population. Therefore we conducted a
series of experiments in which lymph node cells and spleen
cells were used in the CRA, while cells from the same lymph
node poo1 were assayed in the [‘25IJIUdRrelease assay. As
noted in Table 4, the activities of lymph node cells and
spleen cells in the CRA at different times after injection of
MSV were very close in most cases. Although quantitative
differences were observed in some cases strong reactivity in
the [‘25I]IUdR release assay was always accompanied by
strong cytotoxicity in CRA, and weak [125IJIUdR release
assay reactivity was accompanied by weak CRA reactivity,
regardless of the source of lymphocytes.
Table 5 shows the results in [‘25I]IUdRrelease assay and
MCA at various times after MSV inoculation. Nine paired
assays were performed, utilizing lymphocytes from the same
pool of cells for a more direct comparison. Preliminary
2604
I5
spleens
run
individually
in CRA.
experiments with the MCA had shown that at a cell ratio of
1000: 1, no activity was found. However, by increasing the
cell ratio to 2000 to 3000: 1, some significant activity was
obtained. The general pattern of reactivity in the MCA was
very similar to that observed in the CRA (Table 4; Ref. 23)
and in the [‘25I]IUdR release assay (Chart 3). Significant
cytotoxicity occurred during peak tumor growth, 10 to 15
days post-MSV injection, with a subsequent drop in activity
by 20 days to nonsignificant
levels. Occasionally,
we
observed positive reactivity at later time points (e.g., 31
days post-MSV in Table 5), but most mice tested after 20
days gave no significant cytotoxicity. The rapid drop in
cytotoxicity at 20 days in the MCA was not seen in the
[‘25I]IUdRrelease assay or in the CRA and may reflect the
lower sensitivity of the MCA.
Effect of Various Treatments on Cell-mediated Cytotoxic
ity. Lymph node cells were taken from mice 13 days after
MSV inoculation, treated with anti-O antibody plus comple
CANCER RESEARCH
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II
Cel/-mediated
ment, and assayed for residual cytotoxicity in all 3 assays
(Table 6). Treatment with anti-O resulted in almost complete
loss of reactivity in the CRA, as previously reported (17),
and had similar effects on cytotoxicity in the [‘25I]IUdR
release assay and the MCA.
As macrophages have been shown to exert a cytotoxic
effect in some long-term assays, lymph node cells from
MSV-immune
animals at peak reactivity (13 days after
inoculation) were treated with carbonyl iron and then tested
for cytotoxic reactivity (Table 7). In all 3 assays there was
no significant decrease in the cytotoxicity. In fact, in the
are not
Response
to MS V
IUdR release assay, were very similar to those reported
earlier with CRA (23, 26). Reactivity usually appeared
around Day 10 after MSV injection, with a sharp rise to
peak cytotoxicity at 13 to 14 days. In 2 experiments,
reactivity was also present at 3 and 4 days. Subsequently,
the activity rapidly declined so that by Day 20 there was a
low level of reactivity (4 to 8%), which persisted for over 100
days.
Considerable variation was evident in the cytotoxic values
reported for any one time point, e.g., on Day 14 the values
[‘26IJIUdR
releaseassay,therewasa significantincreasein
the level of reactivity, indicating that macrophages
required for the observed cytotoxic activity.
Immune
Comparison ofMCA
Table 5
with [‘2'IIJL'dRrelease assay at various times at Eer
inoculation with MS V
Lymph node cells from mice immunized with MSV were tested at
various times after immunization for their reactivity against MSB target
cells in both the 1―IIIUdR relea@ assay and the MCA. Values for the 2
assays recorded on the same line were obtained with the same cell pools.
DISCUSSION
We have demonstrated
that B6 mice inoculated with
MSV develop cellular immunity against M SV-associated
antigens, which can be measured by the release of [125I]@
lUdR from labeled MSV-transformed
target cells. The
kinetics of the immune response, as measured by [125I]@
release
Days after
MSV
assay
inoculationTumor
1000:1)2000:1―3000:15—@@I0.6c31.0—10.77—7.23.573d10+28.04Ã
statusaMCA[‘25IJIUdR
(500
Table 4
Cs'totoxicit, of li'mph node cells in the I 1251J1L!dRrelease assay and the
CRA as compared to the reactivitt ofspleen cells in the CRA
Pooled lymph node cells and/or pooled spleen cells from the same
animal were tested at various times after immunization with MSV in the
L2SIUdR release assay against MSB cells (lymph node cells only) and in the
CRA against RBL-5 cells (spleen cells and lymph node cells).
cytotoxicity[‘25l]lUdRrelease
Daysafter
assay in lymph
MSV
cells1323.7
inoculation%
node cellsCRALymph
node cellsSpleen
±0.2b15.8
0.4b1433.0±
±l.3a.
b14.8
±0.2'39Ø@Ø4b1533.9 1.6'55.1
0.3'20—3.8± ±0.9'33.3
±Ø5b16.8
1.63.9±0.2―33@Ø3b218.7
0.2―22—5.7±±1.3'8.8
±Ø3@)9.2
±0.7'2714.8
1.010.4±0.6'5.8
0.5'348.9±
±0.4'9.0
±2.5'7.8
l.5b6.8±0.4b6.4±0.4'36—3.1
±0.5―4113.7±2.9―6.2±0.2b6.9±0.3―
±2.07.2±0.5―7.5
±
±
±
±
a Palpable
tumor.
+:
b Lymphocyte:target
a Mean
± SE.
b Significant
cytotoxic
reactivity
above
the
control
(p
<
0.05).
C Percentage
of
d Significant
cytotoxicity
cytotoxicity
no
tumor,
cell
ratio.
—.
above
above
normal
the
control.
control
(p
<
0.05).
Table 6
Treatment of effector populations with anti-O antibody and complement
Lymph node cells from mice given injections 13 days earlier with MSV were treated with anti-O antibody
subsequently tested for residual cytotoxic activity.
MCACRA(200:I―)[“IJIUdR
assay(500-1000:1)2000:13000:1Complement
alone40.5
2.2cAnti-O
plus complement3.
a Lymphocyte:target
cell
b Percentage
of
C Significant
cytotoxicity
SEPTEMBER
cytotoxicity
±07@ C41.6
1 ±0.8c3.9
plus complement
and
release
±Ø3c20.4
±0.5c6.9
±5.6c36.0
±5.5—4.0
±
±2.8
ratio.
above
above
the
control
control
(p
± SE.
<
0.05).
1975
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
2605
G.Fossatieta!.
Table 7
oflvmph node allIs with iron and magnet
Effect ofpretreatment
Lymph node cells from mice injected 13 days earlier with MSV were treated with carbonyl iron and
a magnet. The remaining
cells, which did not ingest iron, were then tested for functional
release
CRA (200: 1)@[125IIIUdR
assay (500- 1000: 1)MCA2000:
1Control37.
I ±I .8―C42.3
l.2c51.0±
[email protected]±
a Lymphocyte:target
cell
b Percentage
of
cytotoxicity
C Significant
cytotoxicity
±0.4c22.6
1.0'19.7±7.7c39.8
13000:
±6.4e42.8
±
ratio.
above
above
the
control
the control
± SE.
(p
<
0.05).
ranged from 3.7 to 49.6%. At this time the source of such
variation remains unknown. Some of the differences can
probably be attributed to animal variation, whether due to
different susceptibility to tumor growth, different cellular
immune response, or slightly different amounts of virus in
the inoculum. Variation in the cytotoxic reactivity of
normal lymphocytes could also contribute to differences in
test results, since experimental cytotoxic levels were relative
to the base line produced by normal lymphocytes. We have
observed normal reactivity in the CRA with RBL-5 target
cells (17, 18) or rat lymphoma (C58NT)D target cells (18),
and in the [‘25I]IUdRrelease assay, with human tumor cell
lines (30), and similar observations have been made in other
laboratories (19, 39). In addition, day-to-day differences in
test conditions (e.g., medium, target cells) could be a source
of variation in the observed cytotoxicity values. In the CRA
we have been able to exclude variation due to normal
lymphocyte reactivity by utilizing as a control unlabeled
target cells in place of normal lymphocytes (23), and it has
been possible to eliminate or monitor discrepancies due to
fluctuations in test conditions by using frozen attacker and
frozen target cells, which give consistent cytotoxicity values
from day-to-day (H. T. Holden, R. K. Oldham, R. B.
Herberman, and D. H. Lavrin, manuscript in preparation).
With similar techniques, attempts are now being made to
minimize variation in the [‘25I]IUdR release assay.
Having established the kinetics of the immune response
as measured by the [‘26I]IUdR release assay, it was of
interest to compare these results to those obtained in the
MCA and the CRA. Disparate results between the mi
crocytotoxicity and 51Cr release assays had previously been
reported by other laboratories. Cytotoxicity as measured by
the CRA peaked at 13 to 14 days after MSV injection with a
rapid decline to low levels by Days 20 to 30 in both BALB/c
(22) and B6 animals (23, 26, 33). In contrast it has been
reported that the cellular response after MSV injection as
measured in the MCA, in both B6 and BALB/c animals,
rose rapidly to a peak at 7 to 10 days [with some reports of
activity as early as 1 day after inoculation (34)], dropped to
low or undetectable levels by 12 days (peak tumor size), and
then peaked again at 15 to 20 days with cytotoxicity levels
remaining elevated for at least 30 days (22, 33). Several
differences in the assay conditions could be cited to account
for these discrepancies. The CRA used a 4-hr incubation
period and suspension cells as targets. In contrast, the MCA
utilized a 48-hr incubation period and adherent cells as
2606
activity.
targets. Furthermore, the culture vessels were different, and
many times the source of lymphocytes (spleen cells or lymph
node cells) was not the same.
These studies were initiated to understand better the
relationship between the assays. To do this we tried to set up
a radioisotopic assay that would measure a cell-mediated
immune response to MSV and that would parallel the
microcytotoxicity
test but would be easier to quantitate.
Therefore, attempts were made to establish assay conditions
in the [‘251]IUdRrelease assay that were similar to those of
MCA. Both assays used the same adherent target cells,
similar incubation periods, a common source of attacker
lymphocytes, and a close relationship in attacker:target
cell
ratios (500 to 1000:1 in [‘251]lUdR release assay 2000 to
3000:1 in MCA). This is in contrast to CRA where a com
pletely different, nonadhrent cell was utilized as the tar
get and there was a shorter incubation period, a lower
attacker:target
cell ratio, and usually a different source of
attacker lymphocytes.
In ourstudiesreportedhere,similarresultswereobtained
in all 3 assays, with peak reactivity occurring at 14 days
post-MSV injection and a subsequent decrease to low levels
thereafter. Low-level activity persisted in the CRA and
[‘25I]IUdR
releaseassaybut not in the MCA, reflecting
perhaps a difference in sensitivity of the assays. The
[125I]IUdR release assay compared favorably to both the
MCA and the CRA with regard to time of appearance of
activity and pattern ofreactivity. In addition, the MCA and
the CRA correlated closely. In contrast to previous reports
(22, 23), activity was found in the MCA at the time of peak
tumor size, while little cytotoxicity was detected beyond 20
to 21 days post-MSV.
Unfortunately,
the paired MCA and [‘25I]IUdR release
assays (Table 5) did not always show correlative results, and
the reason for this is unclear. One explanation might be that
variation in assay conditions were responsible for the
differences. Although the assays were set up under very
similar conditions, there are intrinsic differences that could
contribute to variations in one assay and not in another.
Until assay conditions can be controlled or standardized to
assure uniformity, such discrepancies will probably con
tinue to be a problem.
Not only were the patterns of reactivity similar in these
cytotoxicity tests, but the effector cells appeared to be
similar. Cytotoxic reactivity in all 3 assays was T-cell
dependent, as it was removed after treatment of the effector
CANCER
RESEARCH
VOL. 35
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
Cell-mediated
cells with anti-O antibody and complement, but not depend
ent on macrophages, being unaffected by treatment by the
iron-magnet technique. These finidngs are different from
those of Plata et a!. (33 and Lamon et a!. (22) in the MCA
and from those of Seeger et a!. (38) in the [‘25IJIUdR
release assay. Previously, T-cells and non-T-cells were
found to contribute to both peaks of cytotoxicity in the
MCA, and the subsequent, persistent reactivity was as
cribed to non-T-cells (22, 23). Similarily, non-T-cell killing
was found by Seegar et a!. (38) in the [‘25IJIUdR release
assay, although this activity was attributed to adherent cells,
whereas the cytotoxicity in the MCA was thought to be
mediated by B-cells (22, 23).
The reasons for these major differences from previous
reports are not clear at this time. Many differences in the
experimental
protocols can be cited. (a) Different virus
preparations were used; however, they were all derived from
a common
source. (b) Although
some workers used
BALB/c animals for their investigations our studies were
performed with B6 animals. However, Plata et a!. (33) also
used B6 mice, and strain differences of the effector cells
therefore were not a major factor. (c) The MCA can mea
sure cytolysis as well as cytostasis, while the CRA and
measure only cytolysis. In this study, the pattern of
reactivity as measured by the MCA was similar to that of
the CRA and the [‘25I]IUdR release assay, suggesting
that cytolysis was the main effect we measured by the
MCA. It is possible that, in other laboratories, cytostasis
may be a more important factor in MCA results. (d) One
parameter
of the assays that was distinct from one
laboratory to another was the target cells used. Although
the cells were transformed by similar viruses (murine
leukemia virus or MSV), it cannot be
of the observed reactivity was against
induced by the transforming
agent.
specificity are becoming increasingly
as we
become
more
aware
of the
concluded that all
the same antigens
Determinations
of
difficult to make,
complex
variety
of sur
face antigens that tumor cells possess. As shown by Her
berman et a!. (16) with the CRA, spleen cells from MSV
injected B6 animals were specifically reactive against
tumor cells containing mouse endogenous virus-associated
surface antigen and not (as would be expected) against
tumor cells containing only MSV or murine leukemia
virus antigens. The concordant results that we obtained
in the 3 different assays suggest but do not prove that the
antigens detected by all the tests were the same. Additional
testing against other target cells would be necessary to
answer
this
question.
With regard to the last possibility discussed above, we
have performed some tests that address themselves to the
question of specificity. The MSB target cell used in the
[ 125I]IUdR
release
assay
was
a
syngeneic
MSV-trans
formed cell line. Initial testing indicated that this cell line
was a more suitable target than the allogeneic line,
MSC. The MSB line might be more susceptible to lysis
than the MSC cells [although Seeger et a!. (38) success
fully utilized MSC in a similar assay] or alternately B6 im
munized animals may have recognized antigens on the
MSB cell that were not found on the allogeneic cell line.
Evidence supporting this difference in antigens on MSB
SEPTEMBER
1975
Immune
Response
to MS V
and MSC has been obtained in the CRA (16). Specificity
was also tested utilizing an unrelated target cell. Lympho
cytes from animals given injections of MSV 14 days
earlier failed to kill [‘25I]IUdR-labeled P815 ascites
cells. Similar results were obtained in the CRA. Con
versely, in both the CRA and the [‘25I]IUdR release
assay lymphocytes from P815-immunized animals had
strong
reactivity
reactivity
on
MSB
on P815 ascites cells and only a slight
targets.
As mentioned above, MSV spleen cells are cytotoxic in
the CRA for cells carrying mouse endogenous
virus
associated antigen. P81 5 cells express this antigen after
several
passages
in
vitro
and
are
then
sensitive
to
the
cytotoxic action of MSV lymphocytes (16). When the P815
tissue culture cells were used as a target in [1251]IUdR
release assay with lymphocytes from 14-day MSV animals,
they were positive while P815 ascites cells were not. This
parallel testing, shows that the 2 assays could be measuring
reactivity against common antigens related to mouse endog
enous type C virus expression. However, we cannot con
dude that this is the only reactivity that is being detected,
since reactivity against more than I antigenic specificity
could be generated by the inoculation of MSV into mice.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Holger Kirchner for performing
carbonyl iron-magnet separations.
the
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CANCER RESEARCH
VOL. 35
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
Evaluation of the Cell-mediated Immune Response to Murine
Sarcoma Virus by [ 125I]Iododeoxyuridine Assay and
Comparison with Chromium 51 and Microcytotoxicity Assays
Giuseppe Fossati, Howard T. Holden and Ronald B. Herberman
Cancer Res 1975;35:2600-2608.
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