ICANCER RESEARCH 53. 1921-1928. April 15. 1993]
Enhanced Immunosuppressive
Activity Associated with Metastatic Lymphoma Cells1
Weimin Hao, Thomas L. McDonald, Kenneth W. Brunson, and Shantaram S. Joshi2
Departments of Cell Biology and Anatomy ¡W.H., S. S. J.I and Pathology and Microbiology IT. L M.I. University of Nebraska Medical Center. Omaha. Nebraska 68Ì98-63V5.
and Pittsburgh Cancer Institute. Pittsburgh. Pennsylvania 15213 ¡K.W. B.I
ABSTRACT
Earlier reports from our laboratory showed that Abelson virus-in
duced, highly malignant and liver metastatic RAW117-H10cells, but not
the parental, less metastatic RAW117-1»
cells, inhibited both T-cell and
B-cell mitogen-induced proliferation of syngeneic normal murine spleen
cells. Similar inhibition was also noted when K\\V1I7-IIIO cell surface
molecules extracted with butano! were used instead of whole tumor cells.
In this report we describe the suppressive properties of the butanolextracted RAW117-111((cell surface molecules on other immune functions
and the isolation/purification of a molecule from RAW117-H10 cell bu
tano! extract which shows inhibitory activity. The immunosuppressive
molecules also inhibit natural killer cell-mediated cytotoxicity, lymphokine-activated killer cell-mediated cytotoxicity, and bone marrow colonyforming unit-granulocyte-macrophage colony formation, but not colonyforming unit-fibroblast colony formation. The suppressive molecules
inhibit interleukin 2 production by the T-lymphocytes. One of the mole
cules responsible for some of the immunosuppressive activity has been
isolated and purified from butano!extracts of the metastatic RAW117-H10
cells by preparative isoelectrofocusing techniques. The suppressive mole
cule has an isoelectric point of 4.3 with an approximate molecular weight
of 70,000. Metastatic RAW117-1110lymphoma cells therefore express im
munosuppressive molecules, which may facilitate their growth and metas
tasis i/i vivo.
INTRODUCTION
Immune dysfunction is often observed in advanced cancer patients
(1, 2) and tumor-bearing animals (3). Bast (4) described a selective
action of tumor cells on various components of the immune system
that results in host immunosuppression. Cole el al. (5) reported de
fective T-cell function in patients with established malignancies. Nat
ural killer cells, which are known to play a major role in the antitumor
response, are also known to be affected during tumor progression.
Tursz et al. (6) showed a low NK3 activity in patients with malignant
lymphoma. Badger et al. ( 1) fractionated immunosuppressive factors
from tumor ascites fluid from patients using an in vitro plaque assay
and demonstrated a nontoxic dose-dependent suppression of DNA and
protein synthesis by PHA-stimulated human peripheral blood lym
phocytes by these factors. Control ascites fluid from noncancer pa
tients did not show such an effect. Tumor cell-derived products have
been shown to suppress the immune response (7-14). Ebert et al. (7)
isolated and purified an immunosuppressive molecule from a human
colonie adenocarcinoma cell line HT-29. All of these studies com
pared the effects of tumor cells to normal cells and have not compared
the primary tumor to its metastatic tumor or low-malignancy tumor to
derived highly malignant tumors.
Increased immunosuppression by metastatic tumor cells has been
demonstrated by us (15-17) and by others (18-20). Lovett et al. (18)
Received 12/26/91; accepted 2/11/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by American Cancer Society Institutional Grant 165 and a Grants-in-Aid
Research Award from the national headquarters of Sigma Xi. the Scientific Society.
- To whom requests for reprints should be addressed, at Department of Cell Biology
and Anatomy. University of Nebraska Medical Center. 600 S. 42nd Street, Omaha. NE
68198-6395.
'The abbreviations used are: NK cell, natural killer cell; LAK cell, lymphokine
activated killer cell; PHA. phytohemagglutinin; IL-2, interleukin 2; CFU-F, colony form
ing units-fibroblast; CFU-GM. colony-forming units-granulocyte-macrophage;
CTLL.
cytotoxic T-lymphocyte line; FeLV, feline leukemia virus.
demonstrated that levels of immunosuppression correlated with the
metastatic potential of metastatic variant fibrosarcoma cells. The pre
cise molecular nature of these factors which were responsible for the
immunosuppression were not studied by these investigators. In this
report, we described some of the suppressive properties of the butanol-extracted cell surface molecules from highly malignant and met
astatic RAW117-H10 lymphoma cells. A molecule with suppressive
activity has been identified, isolated, and purified from these cells.
MATERIALS
AND METHODS
Cell Culture. The RAW117-P,RAW117-H10. andYAC-1lymphomacells
were maintained in 60 10-mm Petri dishes (Falcon 1007; Falcon Plastics,
Oxnard, CA) in 5 ml of RPMI 1640containing 10%fetal calf serum (HyClone.
Logan, UT), penicillin (100 units/ml), streptomycin (100 |jg/ml), and
L-glutamine (2 HIM).This medium was termed RF10 medium and is so de
scribed in the following text.
Preparation of Spleen Cell Suspensions. Spleen cell suspensions were
prepared by disrupting the spleens from BALB/c mice by repeated aspiration
with a 1-ml tuberculin syringe without a needle. The cell suspension was
transferred to a conical tube and allowed to settle for 5 min. The supernatant
was transferred to another conical tube and centrifuged at 200 x g for IOmin.
To lyse the red blood cells, the spleen cell pellets were resuspended in an
appropriate volume of ammonium chloride in Tris buffer with a pH of 7.2
(ACT) for 5 min at room temperature. After 5 min, the lytic action of the ACT
solution was neutralized with an equal volume of RF10 medium. The cells
were then centrifuged, washed, and resuspended in RF10 medium for counting
and for further analyses.
Butano! Extraction of Cell Surface Molecules. A modified method of
LeGrue (21) was used to extract the cell surface molecules from RAW117-P.
RAW117-H10 lymphoma, and normal spleen cells using n-butanol. To deter
mine the optimal amount, RAWI17 lymphoma cells were exposed to varying
concentrations of n-butanol. and the optimal concentration, which did not alter
the viability of the treated lymphoma cells, was determined and used for
subsequent extractions. To extract the cell surface molecules, 2 x 10" viable
cells were treated with 2 ml of 2.5% butanol in phosphate-buffered saline for
5 min at room temperature. Cells were centrifuged at 800 rpm for 10 min, and
the supernatant was collected. The supernatant was then centrifuged to remove
the cellular particles at 105,000 x g for 1 h using a Beckman L8-M ultracen
trifuge with a Ti 80 rotor. The supernatant, containing the cell surface mole
cules, was dialyzed against phosphate-buffered saline to remove the butanol
and then used for further analyses. The protein concentration of the butanol
extracts was determined by the method of Bradford (22) using reagents ob
tained from BioRad Laboratories (Richmond. CA).
Natural Killer Cell-mediated Cytotoxicity Assay. Spleencells fromnor
mal BALB/c mice obtained as described above were used as NK effector cells.
The NK cell-mediated cytotoxicity assays were performed as reported previ
ously (23). YAC-1 lymphoma cells were used as target cells in the assay. Five
million YAC-1 target cells were incubated in 100 uCi of MCr (as sodium
enrómate;Amersham, Arlington Heights, IL) at 37°Cfor 45 min. The labeled
cells were washed twice with RFIO medium and adjusted to a concentration of
2.5 X IO5cells/ml. The NK cell-mediated cytotoxicity assay was performed
with or without the butanol-extracted molecules from the metastatic RAW117H10 cells present at the following effectortarget cell ratios: 100:1;50:1; 25:1;
12.5:1.Similar concentrations of butanol-extractedcell surface molecules from
normal BALB/c spleen cells and RAW117-Pcells were used as controls. Cells
were incubated in round-bottomed microtiter plates (Corning Plastics,
Corning. NY) for 4 h at 37°C.Spontaneous release and total release controls
were used for each experiment. Target tumor cells labeled with "Cr and
cultured without the effector cells were used to obtain the spontaneous release
values. Target tumor cells labeled with "Grand lysed with Triton X-100 were
1921
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LYMPHOMA-ASSOCIATED
IMMINORLGI'LATORY
used to obtain the total release value. Triplicate samples were used for each
experimental point. The supernatants were harvested using a Titertech super
natant harvester. "Cr release was measured using a gamma counter, and
used as controls. The culture supernatants were collected and used as the
source of IL-2 for the growth of CTLL-2 cells. The CTLL-2 cells were
obtained from Dr. Mario Stevenson. The CTLL-2 cell line is a cloned cell line
of T-cell origin, which is dependent on IL-2 for its growth. The CTLL-2 cells
percentage cytotoxicity was calculated according to the following formula:
were grown in growth medium supplemented with supernatants containing
IL-2 for a period of 24 h. Recombinant human IL-2 was used as one of the
positive controls for the growth of the CTLL-2. Supernatant obtained from
control flasks cultured without butanol-extracted molecules were used as the
positive growth supernatant. To test if the butanol-extracted molecules from the
metastatic RAW117-H10 cells have a direct antiproliferative effect on the
CTLL-2 cells rather than on the production of IL-2 by the T-cells, CTLL-2 cells
Test cpm - spontaneous cpm
% of lysis =
—¿x 100
Total cpm - spontaneous cpm
NK:Target Cell Conjugate Formation Assay. The effects of the butanolextracted molecules on conjugate-forming ability between NK effector cells
and NK-sensitive target tumor cells was tested, with and without the presence
of butanol-extracted molecules from the metastatic RAW117-H10 cells, ac
cording to the method described by Laybourn et al. (24). Butanol-extracted cell
surface molecules from normal BALB/c spleen cells and RAW 117-P cells were
used as controls. One hundred ul of spleen cell suspension containing 2 x 10'
were cultured with a control supernatant (collected from spleen cells stimulated
with PHA) for 20 h in the presence of butanol extracts of the RAW117-H10
cells and then pulsed by [3H]thymidine. The cells were harvested using an
cells were mixed with lOOul of NK-sensitive RAW 117-P cells containing 1 X
IO5 cells. The mixture was incubated in a glass tube at 37°Cfor 5 min and then
centrifuged for 5 min at 500 X g. The solution was resuspended gently to
disrupt the nonspecific binding. The number of conjugates formed was counted
under the microscope using a hemocytometer. At least 200 target cells/sample
were counted. A conjugate was defined as a target tumor cell attached with one
or more effector cells (24). The percentage of conjugates formed was calcu
lated using the following formula:
% of conjugates formed =
Number of conjugates
Total number of target cells counted
Generation of LAK Cells and LAK Cell-mediated
x 100
automatic cell harvester and counted for radioactivity using a Beckman liquid
scintillation counter (model LB 5180).
In Vitro Mitogen Assay. A suspension of normal BALB/c spleen cells was
obtained as described previously and adjusted to a concentration of 2 X IO6
cells/ml. Two hundred thousand cells were plated into each well of a 96-well
flat-bottomed microtiter plate (Corning Plastics). RF10 medium containing
Con-A ( 100 ul; 5 ug/ml) was added to each well, except for the "medium only"
control wells, to which only spleen cells and 100 pi of medium were added.
Two different concentrations of butanol extracts from the parental RAW 117-P
and the metastatic RAW117-H10 cells were added to triplicate wells. The
cultures were incubated for 72 h at 37°Cwith 5% CO: and 95% air. To evaluate
the synthesis of DNA, these cultures were pulsed with 1 uCi of ['H]thymidine
(NEN Research Products. Boston. MA) 54 h after initiation of culture. The
cells were then incubated for another 18 h and harvested onto glass fiber filter
paper discs using a Skatron cell harvester. The filter paper discs were dried, and
the radioactivity was counted using a Beckman liquid scintillation counter
(model LB 5180) after putting each disc into a 3-ml plastic tube containing
Cytotoxicity Assay.
Spleen cells were obtained from normal BALB/c mice as described above.
Then 2.5 X IO7 cells were incubated in 20 ml of RF10 medium with 1000
units/ml of recombinant human ¡merleukin 2 (a generous gift from Cetus
Corporation. Emeryville. CA) and 2 x 10~5 M 2-mercaptoethanol for 3 days.
scintillation solution.
Preparative Isoelectrofocusing
of Butanol-extracted
Molecules. The
crude molecules obtained from the metastatic RAW117-H10 cells using bu
tanol were fractionated using a BioRad "Rotofor" preparative isoelectrofocus-
The cells were washed twice with RF10 medium and then used as LAK cells.
The LAK assays were performed with and without butanol-extracted mole
cules from the metastatic RAW117-H10 cells, with butanol-extracted mole
cules from normal BALB/c spleen cells and RAW 117-P cells as controls. The
remaining procedures for the LAK cell-mediated cytotoxicity assay were iden
tical to those of the NK cell-mediated cytotoxicity assay.
Bone Marrow CFU-GM Assay. The effects of butanol-extracted mole
cules on bone marrow CFU-GM colony formation were assayed using the
semisolid agar method described by Metcalf et al. (25). For CFU-GM assays,
ing unit. The butanol extracts were dialyzed overnight against distilled water
containing 0.005% n-octylglucoside. The presence of n-octylglucoside was to
prevent mycellae/aggregate formation between the cell surface molecules.
Some of the molecules extracted were precipitated during this dialysis against
distilled water. Subsequent analysis of the precipitate showed that it did not
contain a significant inhibitory activity. The dialyzed molecules were then
electrofocused in a 1% solution of selected ampholyte (pH 3-11 ; BioRad) for
3-4 h. The electrofocused samples were collected into 20 sharply defined
fractions based on the isoelectric points of the individual fractions (the rotating
Rotofor chamber is partitioned into 20 compartments, each separated from the
other by a porous membrane and cooled by a water-filled ceramic finger). The
normal BALB/c bone marrow cells were obtained from mouse femurs. Bone
marrow cells (2 X 10s) were suspended in McCoy's medium containing 0.3%
melted agar and 0.1 ml of KLN-205 culture supernatant as a source of colonystimulating factor (26). This mixture was plated in 35-mm plastic Petri dishes
with or without butanol-extracted molecules. A similar concentration of bu
tanol-extracted cell surface molecules from RAW 117-P cells was used as the
control. The plates were incubated in 5% CO2 in a humidified atmosphere at
37°C.The colonies were stained after 7 days with Wright's stain and scored
using a Bélicocolony counter. Aggregates larger than 50 cells were counted as
a colony.
Bone Marrow CFU-F Assay. CFU-F assays were performed according to
the method described by Wellman el al. (27). Bone marrow cells (5 x IO5)
were cultured in 35-mm plastic Petri dishes containing 1 ml of RPMI 1640
with 25% fetal calf serum, penicillin (100 U/ml). streptomycin (100 ug/ml).
and L-glutamine (2 ITIM).Culture dishes were incubated for 8 days at 37°Cin
5% CO2 in a humidified atmosphere with or without butanol-extracted mole
cules from the metastatic RAW117-H10 cells. A similar concentration of cell
surface extracts measured as protein concentration from RAW 117-P lymphoma
cells was used as a control. Media were removed from the cultures on day 8,
cultures were stained using Wright's-Giemsa stain, and the number of colonies
was counted.
IL-2 Production Assay. These experiments were carried out according to
a modified method of Eben et al. (7). Spleen cells from normal BALB/c mice
were collected as described above. Twenty million spleen cells/flask were
cultured for 2 days in RF10 medium in the presence of PHA at a concentration
of 1 ug/ml, 1 X IO"5 M 2-mercaptoethanol. with or without butanol-extracted
molecules from the metastatic RAW117-H10 cells. The butanol-extracted cell
surface molecules from normal BALB/c spleen cells and RAW 117-P cells were
MOLECULE
pH of each fraction after electrofocusing was determined using a pH meter.
Each fraction obtained was then dialyzed against phosphate-buffered saline
with four to five changes, to remove the ampholyte, and used for its inhibitory
properties in an in vitro mitogen assay as described previously. The inhibitory
activity of fraction 7. which showed an inhibition of mitogen response, was
repeated along with a fraction with a similar pi value from the parental
RAW! 17-P cell butanol extracts using an identical mitogen assay.
Electrophoretic Analysis of Purified Molecules. This active fraction ob
tained by preparative isoelectrofocusing analysis, which showed a significant
inhibition of mitogen-induced proliferation of normal spleen cells, was ana
lyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis according
to the method of Laemmli (28). Samples (5 and 8 ug) from the active fraction
as well as molecular weight markers were loaded on lanes on a 10% gel and
electrophoresed. The gel was then stained with 0.05% Coomassie blue in 50%
methanol and destained with destaining solution (25% methanol, 10% acetic
acid, and 65% water).
Western Blotting Analysis. Fraction 7. which showed inhibitory activity,
was electrophoresed using a sodium dodecyl sulfate-polyacrylamide gel elec
trophoresis system as described above. The Western blotting analysis was
performed according to the method of Lin and Kasamatsu (29). The electrophoretic gels as well as the filter pad and the filter paper that were to be used
in the blotting process were left in blotting buffer for at least 30 min to be
equilibrated. The molecules were transferred electrophoretically onto a nylon
1922
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LYMPHOMA-ASSOCIATED
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MOLECULE
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membrane soaked in blotting buffer for 30 min prior to blotting using the
BioRad Transblot system. The nylon membrane containing molecules from
fraction 7 was soaked in phosphate-buffered saline with 0.5% bovine serum
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albumin for 30 min and incubated with polyclonal antibodies prepared in
rabbits using a M, 70,000 glycoprotein. other than wild-type viral envelope
gp70, obtained from metastatic RAWI17-H10 cells (23). The filter paper was
washed 3 times using phosphate-buffered saline and incubated with phosphatase-conjugated secondary antibodies (BRL. Rockville. MD) for I h. The
membrane was washed 3 times with phosphate-buffered saline before exposing
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it to the color developing solution containing 50 ul of MgCN (2.0 M)/25 ml of
25 HIMTris buffer (pH of 9.0), 100 ul of 5 mg/ml 5-broma-4-chloro-3-in-
5.a.0-CZI
dodylphosphate and 5 mg nitroblue tetrazolium. The reaction was stopped after
30 min with distilled water.
Antibody Blocking Experiments. The polyclonal antibody which reacted
with a M, 70,000 glycoprotein expressed on metastatic RAW 117-H10 cells was
also used to block the inhibition of mitogen activity of fraction 7 using an in
vitro mitogen assay. Three different concentrations of polyclonal antibodies
(1:100, 1:200, and 1:400) were added into wells containing spleen cells,
mitogen, and fraction 7 molecules. Normal rabbit serum at a concentration of
1:100 was used as control antiserum in this assay.
O
2.5
5.0
2.5
5.0
2.5
5.0
Concentration of Butanol Extracted Molecules (/ig/200
/¿I)
RESULTS
Fig. 2. Graphic representation of the effects of butanol extracts from the highly met
astatic RAWII7-H10. parental RAWI 17-P. and normal BALB/c spleen cells on the
conjugate formation between the NK effector cells from BALB/c mice and the NK
susceptible RAWI 17-P target cells. Values in the figure are means ±SEM of three
separate experiments. The concentrations of extracts indicated in this graph arc the amount
of protein in each test tube in a total volume of 200 ul. *. significant difference between
extracts of normal spleen cells and those of RAWI] 7-P or RAWI 17-H 10 cell groups. T,
significant difference between extracts from RAW 117-P and those from RAW II 7-H 10
groups.
The effects of butanol extracts from the metastatic RAW 117-H 10
cells on NK cell-mediated cytotoxicity were investigated using the in
vitro NK cell-mediated cytotoxicity assay (Fig. 1). Fresh spleen cells
from BALB/c mice were used as NK effector cells, and the NKsusceptible YAC-1 lymphoma cells labeled with 51Cr were used as
target tumor cells. The NK cell-mediated cytotoxicity assays were
performed in the presence of the butanol extracts from RAW 117-H 10
cells, with RAWI 17-P cells and normal BALB/c spleen cells as con
trols. The assays were performed at different effectontarget cell ratios.
In all of these NK cytotoxicity assays, the spontaneous release was
less than 10%. Fig. 1 illustrates the effects of the butanol extracts from
these cells on NK cell-mediated cytotoxicity. The NK cell-mediated
cytotoxicity assays with and without butanol-extracted cell surface
molecules were repeated four times, and the figure represents the
mean values of these experiments. The butanol extracts from
RAWI17-H10 cells at a concentration of 2.5 and 5.0 ug/well were
significantly inhibitory to NK cell-mediated cytotoxicity when com
pared to that with butanol extracts from normal spleen cells. The
butanol extracts from RAWI 17-P cells did not significantly inhibit the
NK cell-mediated cytotoxicity when compared to normal spleen cell
extracts. At the effectontarget ratio of 100:1, the butanol extracts from
RAW117-H10 lymphoma cells were significantly more inhibitory to
NK cell-mediated cytotoxicity than butanol extracts from RAWI 17-P
lymphoma cells (at a concentration of 5 ug/well) (P < 0.025, P < 0.01
for 2.5 and 5 ug/well, respectively).
In order to determine whether the NK-suppressive butanol extracts
from RAWI 17-H 10 lymphoma cells interfered with conjugate forma
tion between NK effector cells and their target cells, butanol extracts
were added to culture medium in a standard conjugate formation
assay. Fig. 2 demonstrates the effects of two different concentrations
(2.5 and 5.0 ug) of butanol-extracted cell surface molecules from
parental RAWI 17-P cells, metastatic RAWI 17-H 10 cells, and normal
BALB/c spleen cells on conjugate formation between effector cells
and target cells. The butanol-extracted molecules from RAWI 17-H 10
cells inhibited the conjugate formation significantly (P < 0.005, for
both 2.5 and 5.0 ug). Butanol-extracted
molecules from the
RAWI 17-P cells also inhibited the conjugate formation, although to a
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Fig. 1. Graphic representation of the effects of butanol extracts from the highly met
astatic RAW1I7-HIO lymphoma cells, parental RAWH7-P cells, and normal BALB/c
spleen cells on NK cell-mediated cytotoxicity. Values in the figure are means ±SEM of
four separate experiments. The concentrations indicated in this figure are the amount of
protein added per well. SPC EXT. spleen cell extracts; RAW/17-P EXT. RAWI 17-P cell
extracts; RAW117-H 10 EXT. RAWI 17-H10 cell extracts. »,significant difference between
extracts of normal spleen cells and those of RAWI 17-P or RAWI 17-H 10 cell groups. T.
significant difference between extracts from RAWI17-Pand those from RAW1I7-HIO
groups.
lesser extent. Results were statistically significant when compared to
normal spleen cell extracts (P < 0.01, P < 0.01 for 2.5 and 5 ug,
respectively). But the inhibition of conjugate formation by the bu
tanol-extracted molecules from metastatic RAWI17-H10 cells was
significantly greater when compared to butanol extracts from parental
RAWI 17-P lymphoma cells (P < 0.05). The values in the figure
represent means ±SEM of three separate experiments.
The effect of butanol-extracted molecules on LAK cell-mediated
cytotoxicity was studied using an in vitro LAK cell-mediated cyto
toxicity assay (Fig. 3). Spleen cells from BALB/c mice were cultured
with recombinant human IL-2 (1000 units/ml) and incubated for 4
days and then used as effector cells in a LAK cell-mediated cytotox
icity assay. The LAK cell-mediated cytotoxicity assays were per
formed in the presence of two different concentrations of butanol
extracts from the metastatic RAWI 17-H 10 cells, parental RAWI 17-P
cells, and normal BALB/c spleen cells. Control assays were per
formed without the butanol extracts, but with appropriate volumes of
medium alone. The LAK cell-mediated cytotoxicity assays were per
formed using various effectontarget cell ratios. Fig. 3 demonstrates
the effects of butanol extracts from metastatic RAW 117-H 10 cells,
parental RAWI 17-P cells, and normal BALB/c spleen cells on LAK
cell-mediated cytotoxicity. Both concentrations (2.5 and 5.0 ug/well)
of the butanol-extracted molecules from the metastatic RAWI 17-H 10
1923
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I.YMPHOMA-ASSOCIATED
IMMUNOREGULATORY
To study the effects of butanol-extracted molecules from the met
astatic RAW 117-H 10 cells on the production of IL-2 by spleen cells,
two concentrations of butanol-extracted molecules were added to the
cultures of spleen cells stimulated with PHA at the initiation of the
culture. The cell surface molecules (6 pg/ml) similarly extracted from
parental RAW 117-P cells and normal BALB/c spleen cells were used
as controls. The supernatants from such cultures were collected and
used to evaluate the growth of the IL-2-dependent CTLL-2 cell line
using a [3H]thymidine uptake method. Fig. 4 shows the percentage of
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Fig. 3. Graphic representation of the effects of butanol extracts from the highly metastatic lymphoma RAW 117-H 10 cells, parental RAW 117-P lymphoma cells, and normal
BALB/c spleen cells on LAK cell-mediated cytoioxicity. Values in the figure are means
±SEM of four separate experiments (except for RAW117-P cells extracts, where n = 3).
The concentrations indicated in this figure are the amount of protein per well. *, signif
icant difference between extracts of normal spleen cells and those of RAW 117-P or
RAWI17-HIO cell groups. V. significant difference between extracts from RAWI17-P
and those from RAW 117-H 10 groups.
Table 1 Effects of butanol-extracied molecules on the colony-fanning
bone marrow progenitor cells (CFU-GM)
of colonies,
CFU-GM"I00.75±6.I7
MOLECULE
ability of mouse
DCS0.05S0.025
Medium-only controls
RAW II 7-P extracts
61.35±16.42
NSrf
RAW117-H10 extractsNo.
62.56±13.03pk
" Mean ±SEM of four separate experiments, 2 x IO5 normal Balb/c mouse bone
marrow cells/dish with 2 ug/dish butanol-extracted molecules added.
h P values between control and RAW117-P extract or RAW117-H10 extract groups.
'' P values between RAW 117-P extract and RAW 117-H 10 extract groups.
d Not significant.
inhibition of the growth of CTLL-2 cells. When compared with the
inhibition by the normal spleen cell extracts, only the metastatic
RAW 117-H 10 cell extracts at a concentration of 6 ug/ml significantly
inhibited CTLL-2 cell growth in vitro (P < 0.005). Neither RAW117H10 extracts nor RAW117-P extracts (6 ug/ml) at lower concentra
tions showed significant inhibition of growth of CTLL-2 cells in vitro.
The inhibition of CTLL-2 growth in vitro by the metastatic RAW 117H10 cell extracts was significant when compared to that of RAW 117-P
cell extracts (P < 0.05 at the higher concentration), indicating the
enhanced inhibitory effects associated with RAW 117-H 10 cells. The
values in the figure are means ±SEM of six separate experiments
(except spleen cell extracts and RAW 117-P extracts, where n = 3). To
examine the possibility that butanol-extracted
molecules from
RAW 117-H 10 cells might be directly inhibiting the growth of CTLL-2
rather than inhibiting the production of IL-2, butanol extracts from
metastatic RAW 117-H 10 cells were added to the CTLL-2 prolifera
tion assay directly with the positive control supernatant (only PHA
and spleen cells were added for the collection of the positive super
natants). The direct addition of butanol-extracted molecules from
Table 2 Effects of the butanol-extracted molecules on ¡hecolony-forming abilities
of the murine bone marrow progenitor cells (CFU-F)
No. of colonies,
CFU-F1
Ph
P1
cells significantly inhibited LAK cell-mediated cytotoxicity when
Medium-only
controls
32.90
±6.34
compared to butanol extracts from normal spleen cells. Extracts from
NS''
RAW117-Pextarcts
23.65 ±3.93
the parental RAW117-P cells did not significantly inhibit LAK cellRAWI17-H10 extracts
18.65 + 6.76
NS
NS
mediated cytotoxicity when compared to normal spleen cell extracts.
" Mean ±SEM of four separate experiments. 5 x IO5 normal Balb/c mouse bone
When the inhibition of LAK cell-mediated cytotoxicity by metastatic
marrow cells/dish with 2 pg/dish butanol-extracted molecules added.
* P values between control and RAW117-P extract or RAW] 17-H10 extract groups.
RAW117-H10 cell extracts was compared to that of parental
' P values between RAW 117-P extract and R AW II 7-H 10 extract groups.
RAW 117-P cell extracts, the RAW 117-H 10 cell extracts were signif
^ Not significant.
icantly more inhibitory (P < 0.05). Such a significant inhibition was
not seen with either butanol extracts from RAW 117-P cells or normal
BALB/c spleen cells. The values in the figure represent means ±SEM
60 T
ES RAW117-H10 EXT.
CDSPC EXT.
IZZ3 RAW117-P EXT.
of four separate experiments.
50-The effects of butanol-extracted cell surface molecules from the
metastatic RAW1I7-H10 and the parental RAW117-P cells was also
m 40302010-A..tested on bone marrow colony formation as measured by CFU-GM
and CFU-F assays. The results indicate that the cell surface molecules
from both RAW 117-H 10 cells and RAW 117-P cells significantly sup
pressed the colony-forming abilities of syngeneic BALB/c bone mar
'y
'/
row cells in CFU-GM assays (Table 1) (P < 0.05 for RAW117-P
u
/
oc.
f\Õi\y//////%1\I
/•
extracts; P < 0.025 for RAW117-H10 extracts) when compared to
medium-only controls. However, there was no difference between the
inhibition of the cell extracts from the two cell lines. The values in the
table are means ±SEM of four separate experiments.
6636
Table 2 demonstrates the effects of butanol-extracted cell surface
Concentration
of Butanol Extracted Molecules
molecules from RAW 117-H 10 cells and RAW 117-P cells on bone
Fig. 4. Graphic representation of the effects of butanol extracts from the highly met
astatic RAW 117-H 10 lymphoma cells, parental RAW 117-P lymphoma cells, and normal
marrow CFU-F colony formation. Butanol-extracted cell surface mol
BALB/c spleen cells on the production of IL-2 by murine T-cells. as measured by the
ecules from both the RAW 117-H 10 cells and RAW 117-P cells de
growth of the IL-2-dependent CTLL-2 cell line. Values shown in the figure are the
creased the CFU-F colony formation by syngeneic BALB/c bone
percentage of inhibition demonstrated by supernatants containing various butanol extracts.
Values are means ±SEM of six separate experiments (except normal spleen extracts and
marrow cells when compared to controls. This apparent decrease was
RAW117-P extracts, where n = 3). *. significant difference between extracts of normal
not statistically significant. The values in the table are means ±SEM
spleen cells and those of RAW 117-P or RAW 117-H 10 cell groups. T, significant differ
ence between extracts from RAW 117-P and those of RAW 117-H 10 groups.
of four separate experiments.
1924
[i|7
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LYMPHOMA-ASSOCIATED
IMML'NOREGULATORY
RAW117-H10 cells in CTLL-2 assays did not significantly alter the
CTLL-2 growth when compared with controls (data not shown).
Before purifying or isolating the molecule(s) responsible for the
inhibitory activity, the butanol extracts from the parental RAW117-P
and the metastatic RAW117-H10 cells were tested for their inhibitory
activity on mitogen-induced proliferation of normal BALB/c spleen
cells. Table 3 demonstrates the results of these experiments. When
compared to controls, both concentrations of butanol extract from
metastatic RAW117-H10 cells significantly inhibited the mitogen re
sponse (P < 0.0005 for both 2.5 and 5 ug/well). Only the higher
mitogen response (P < 0.0005, for both 2.5 ug/well and 5 ug/well).
Only the higher concentration of butanol extract from the parental
RAW117-P cells significantly inhibited the mitogen response (P <
0.005). The butanol extracts from RAW117-H10 cells demonstrate a
significant inhibitory effect on mitogen-induced proliferation of nor
mal spleen cells when compared to RAW117-P cell extracts (P <
0.0005, P < 0.005 for 2.5 and 5 ug/well, respectively). These exper
iments were repeated 3 times; the table represents the mean of these
separate experiments.
The crude butanol extracts from RAW117-H10 cells that contain
suppressive activities were fractionated using the Rotofor preparative
isoelectrofocusing unit (BioRad). The suppressive activity of each
fraction was determined using an in vitro mitogen assay. Fig. 5 rep
resents results showing the inhibitory activity and pH values of each
fraction. Fraction 7 was found to be the active fraction, which dem
onstrated a significant suppressive effect on mitogen-induced prolif
eration of normal spleen cells. The isoelectric point of fraction 7 was
4.3 (Fig. 5).
The effects of fraction 7, along with a fraction with a similar pi
value obtained from the parental RAW117-P cell extracts, on the
mitogen-induced proliferation of BALB/c spleen cells are shown in
able 4. A significant inhibitory effect by fraction 7 from RAW 117Table 3 The effects of the crude butanol extracts on mitogen-induced proliferation
of normal spleen cells
MOLECULE
Table 4 Effects of the fraction 7 molecules from the RAW117-HIO lymphoma cells
on concanavalin A-induced proliferation of normal spleen cells
Spleen
Spleen
Spleen
Spleen
cells
cells
cells
cells
only
±380
+ concanavalin A
137,898 ±7,037
+ RAW 117-P extracts1'
124,957 ±7,251
+ concanavalin A + fraction I1cpm"1.123
<0.0005PcS0.0005
8,102 ±172P"NSf
" Values in the table are mean ±SEM of three separate experiments.
h P values between controls and extracts from RAW117-H10 or RAW117-P.
' P values between RAW 117-P extracts and fraction 7 molecules.
'' RAW 117-P cell extract fraction with similar pi value from the isoelectric focusing
analysis (1.25 ug/well).
*"Not significant.
^Fraction 7, 1.25 ug/well.
a
b
cd
200kD
160
93
68
43
25
12
Fig. 6. Photograph of the results of the sodium dodecyl sulfate-polyacrylamide gel
electrophoretic analysis of fraction 7. which showed inhibition of the mitogen response of
normal spleen cells. This active fraction showed a major band at about M, 70,000 (arrow).
Lanes a and b. fraction 7 obtained from preparative isoelectrofocusing. two concentrations
(8 ug) and (5 ug) of Coomassie blue stain; Lanes c and d. molecular weight markers.
±6.083
Control
RAW 117-P EXT. ld
137,326 ±5,899
RAW 117-P EXT. 11
79,985 ±4,979
S0.005
RAW117-HIOEXT. I
40,629 ±2.420
S0.0005
RAW117-H10EXT. IIcpm"133.37529,037 ±3,549P"NS' S0.0005/"<0.0005 S0.005
" Mean ±SEM of three separate experiments.
h P values between controls and extracts from RAW117-HIO or RAWI 17-P.
CP values between extracts from RAWI 17-HIO and RAWII7-P.
rfl, 2.5 ug/well; II, 5.0 ug/well.
**Not statistically significant.
H10 cell extract was observed when compared with the control (P <
0.001). The same fraction with a similar pi value obtained from
RAW117-P cell extracts did not show any significant inhibitory ac
tivity. The same fraction also inhibited the NK cell-mediated cytotoxicity of normal spleen cells against YAC-1 tumor target cells (data not
shown).
In order to further characterize these molecule(s), fraction 7 was
electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel.
The electrophoretic analyses showed that fraction 7 contained a major
^opu.
band of Mr 70,000 and a minor band of A/r 85,000 (Fig. 6). Polyclonal
antibody raised against a purified Mr 70,000 molecule from the met
./-.0'°
ce
astatic RAW117-H10 cells was also reactive with the molecule in
¡¿100.Z
0
D—
fraction 7 as well as crude butanol extracts from RAW117-H10 cells.
St
QThe
polyclonal antibody did not react with the crude extracts from
-(PI
_,1
OD
o,0A
4.3)
normal BALB/c spleen cells, as determined by the Western blotting
-o-5
«
Z
u•0-0-°-°"°"<J\
o
technique (Fig. 7).
I-4
so.5
5
Because fraction 7 contained a major A/r 70,000 molecule and a
y0
Q.an
-3
minor molecule of about Mr 85,000, we performed antibody blocking
vi
_i0¡fJ
-2-1»n assays using mitogen assays as described in the methodology section,
<2O(/.O
i •¿-•~T~
. •¿-•-•"^-y"
i ^•-•-••y
^-(J-11-10-9-8r-7-6
in order to determine which of these molecules had inhibitory activity.
Fig. 8 demonstrates the results of these experiments. The polyclonal
0 1 2 3 4 5 6 7 8 91011121314151617181920
antibodies reacting with the fraction 7 molecule blocked the suppres
FRACTION
NUMBER
sive activity of the fraction 7 molecule to a significant extent. The
Fig. 5. Graphic representation of the pH profile of the 20 fractions from the preparative
blocking was concentration dependent. Among three concentrations of
isoelectrofocusing analysis and the effects of each fraction on mitogen-induced prolifer
polyclonal antibody used, the 1:100 dilution showed maximum block
ation of normal spleen cells. The value in the figure represents the mean of two separate
experiments. O, pH values of each fraction; •¿.
inhibitory activity of each fraction.
ing of the suppressive activity of fraction 7. The 1:200 dilution of
;•'
...
1925
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LYMPHOMA-ASSOCIATED
ABC
IMMUNORI-iOl'LATORY
MOLECULE
variant lymphoma cells probably contain more than one molecule with
inhibitory activity. Subsequent isolation and purification of a molecule
-200 kD responsible for some of this inhibition revealed that the molecule is a
glycoprotein with an approximate molecular weight of 70,000 and an
-116
isoelectric point of 4.3. Although several investigators have reported
- 97
properties of various tumor cell-associated immunoregulatory mole
- 68
- 45
cules, the one which is reported in this study is significantly expressed
only on the highly metastatic/malignant RAW117-HK) cells but not on
_ 31
the low-malignancy/metastatic parental RAW 117-P cells from which
- 21
the metastatic RAW 117-HIO cells were developed by in vivo selection
techniques (16). Although the inhibitory molecules are expressed on
the parental/low metastatic RAW 117-P cells, the amount expressed is
significantly low when compared to metastatic RAW 117-HIO cells. In
this regard there are very few reports on increased immunosuppression by metastatic tumor cells. Lovett et al. (18) demonstrated met
astatic fibrosarcoma-induced immunosuppression. In their studies, the
Fig. 7. Photograph of the Western blotting analysis using monospecific polyclonal
levels of immunosuppression correlated with the metastatic potential
antibody against a Mr 70,000 molecule from the RAW117-H10 cells. Lane A, butanol
of the tumor cells. The molecules responsible for such immunosup
extracts from normal spleen cells; Lane B, fraction 7 obtained from preparative isoelectrofocusing; Lane C, crude butanol extracts from RAW 117-HK) cells; Lane D, molecular
pression by the fibrosarcoma cells have not been isolated and purified.
weight standard markers.
Eben et al. (7) reported the isolation and purification of immunosuppressive molecules from human adenocarcinoma cell line HT-29.
ESFRACTION7
CD SPC EXT. Za WW117-PEXT.
These molecules have a molecular weight of 56.000 and a pi of 7.9.
70 T
•¿
Ab.
m
Normal Serum
Remacle-Bonnet et al. (19) reported the isolation of a M, 70,000
molecule from fresh tumor extracts from esophageal cancer. Similarly.
Z
60-Werkmeister et al. (30) also reported an immunosuppressive molecule
O
with a molecular weight of 70,000 isolated from liposarcoma cells.
l
*> +
Itoh et til. (31) have reported that serum from patients with meta
ï
40-static melanoma, but not from healthy donors, inhibited the induction
of LAK cells by IL-2. Furthermore, Riley et al. (32) reported two
30-distinct inhibitors of LAK cell induction from serum from patients
LJ
U
with advanced cancer. One of these is aggregated IgG but not monomeric IgG, and the other one is a Mr 65,000 molecule distinct from
10-either IgG or serum albumin. The IgG aggregate requires monocyte to
exert inhibition of LAK cell generation. The other molecule appears to
fi
O.t
1:100 1:200 1:400 1:100
O.t
act directly on LAK cell induction. In comparison to these studies, our
Concentration of Butanol Extracts or Fraction 7 (/¿g/well) molecule may be similar to the latter component. However, the im
munosuppression we describe in this paper is associated with highly
Fig. 8. The antibody blocking assay using monospecific polyclonal antibody to block
metastatic lymphoma cells. In their studies, the origin of the M, 65,000
the suppressive activity of the fraction 7 molecules ¡nthe in vitro mitogen assay. Values
in the figure are means ±SEM of three separate experiments. *, significant difference
molecule present in serum from cancer patients is not yet established.
between extracts of normal spleen cells and those of RAW117-P or RAW117-HIO cell
Immunosuppression is known to accompany retroviral infections
groups. T. significant difference between extracts from RAW 117-P and those from
RAW117-HIO groups.
and often precedes the development of cancers in animals infected
with oncogenic viruses (33). The major molecule associated with the
virus-associated immunosuppression are viral envelope protein pl5E.
polyclonal antibody also showed some blocking activity (Fig. 8). Such
The pl5E is a hydrophobic transmembrane protein with an approxi
a blocking of the suppressive activity was not observed with normal
mate molecular weight of 19,000 and is a cleavage product from
rabbit serum (1:100 dilution). The results indicate that the MT70,000
precursor polypeptide with an approximate molecular weight of
molecule might be responsible for the observed suppressive activities.
90,000. The other cleavage product of this precursor polypeptide is the
envelope glycoprotein gp70. The molecule which we describe here
DISCUSSION
has a molecular weight similar to that of the latter molecule. Immu
In the present study we have demonstrated that cell surface mole
nosuppressive effects of the retroviral envelope protein pl5E have
been demonstrated (34-37). Moreover, Cianciolo et al. (34-36) have
cules extracted with butanol from the highly malignant metastatic
RAW117-HIO cells have an inhibitory effect on NK and LAK cellshown that CKS-17, a synthetic peptide homologous to a conserved
region of retroviral envelope protein, inhibited the proliferation of
mediated cytotoxicity, conjugate formation between the effector cells
and their tumor target cells, and CFU-GM and CFU-F colony forma
murine CTL cell line and alloantigen-stimulated murine and human
tion by the bone marrow cells. The inhibition of bone marrow CFU-F
lymphocytes. Furthermore, they have also reported the presence of
pl5E-like protein in human cancerous effusions and in mitogen-transcolonies by the lymphoma cell surface molecules was not statistically
formed human lymphocytes (38). Jacquemin and Strijckmans (39)
significant. The cell surface molecules obtained from the less meta
static parental RAW117-P cells also inhibited conjugate formation
reported that there are elevated plasma levels of pl5E-like glycopro
between NK effector cells and tumor target cells and bone marrow
tein in leukemic patients. In the RAW117 lymphoma system, although
CFU-GF colony-forming ability. However, the cell surface extract
these cells were transformed by Abelson leukemia virus with Moloney
from parental RAW117-P cells did not inhibit other immune function
leukemia virus as a helper virus, the immunosuppressive effects ob
parameters such as LAK cell-mediated cytotoxicity. These results
served are mainly restricted to the highly malignant RAW 117-HIO
indicate that the crude cell surface extracts from these metastatic
cells. However, the amounts of viral envelope glycoprotein gp70 and
1926
D
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1993 American Association for Cancer Research.
LYMPHOMA-ASSOCIATED
IMMUNOREGULATORY
pl5E expressed on the RAW117-H10 cells are known to be less than
that expressed on the parental RAW 117-P cells (40). Therefore, these
wild-type viral products are not likely to be causative agents for the
immunosuppression unless they are present in an altered form on
RAW117-H10 cells. In fact, we have earlier reported increased ex
pression of a M, 70,000 glycoprotein molecule (different from viral
envelope glycoprotein gp70) by the RAW117-H10 cells when com
pared to low-metastatic parental RAW 117-P cells (23). At that time we
did not know that these molecules had an inhibitory effect on the
immune system. It is only recently that we have found that the Mr
70,000 molecules which are expressed more on RAW117-H10 cells
have significant immunosuppressive properties. The classical or wildtype viral envelope glycoprotein gp70 is not the immunosuppressive/
antiproliferative molecule. On the basis of these findings the immu
nosuppressive molecules we have reported here could be an altered
viral envelope protein. This needs to be confirmed by further charac
terization of the molecule.
Recently, a replicative defective variant of feline leukemia virus
which induces a rapid and fatal immunodeficiency syndrome in cats
was reported by Overbaugh et al. (41). Their studies with cloned
viruses also showed that subtle mutational changes in viral envelope
gp70 gene would convert a minimally pathogenic virus into one that
would induce an acute form of immunodeficiency. This mutant viral
product appeared to inhibit mainly T-cell function and bone marrow
progenitor cell function, which resulted in severe immunodeficiency
syndrome in cats (42). Poss et al. (43^-5) reported a significant
difference in the envelope glycoproteins from major pathogenic FeLVFAIDS virus and wild-type FeLV. In both cases, precursor glycopro
tein gp80 and the mature extracellular glycoprotein gp70 from FeLVFAIDS were larger than the wild-type virus. Furthermore, they have
shown that the envelope glycoproteins of the pathogenic variant virus
differ from the apathogenic parent virus in the antigenicity rate of
processing and carbohydrate constituents. These studies on FeLVFAIDS virus and their products demonstrate the highly pathogenic
properties of a mutant virus gene product.
Although the biological significance of the metastatic RAW 117HlO-associated immunosuppression is not clear, these cells seem to
induce host immunosuppression in their host animals. Recently, we
have found that there is a significantly decreased immune response in
syngeneic BALB/c mice bearing RAW117-H10 lymphoma, particu
larly 6 days after tumor transplantation (45). Such a significant de
crease in host immune response was not seen in animals bearing low
malignant/metastatic RAW 117-P cells or in untreated control animals.
Further studies on the immune response of normal BALB/c animals
given injections of purified immunosuppressive molecules are cur
rently in progress.
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ACKNOWLEDGMENTS
We thank Dr. Geoffrey Thiele for providing normal mouse spleen cells and
Roberta Anderson for typing the manuscript.
28.
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Enhanced Immunosuppressive Activity Associated with
Metastatic Lymphoma Cells
Weimin Hao, Thomas L. McDonald, Kenneth W. Brunson, et al.
Cancer Res 1993;53:1921-1928.
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