Humoral enhancement of metastasis : circulating IgG interactions with tumor-bearing lymphocytes
by Cheryl Juline Aslakson
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Microbiology
Montana State University
© Copyright by Cheryl Juline Aslakson (1986)
Abstract:
Using an inbred BD-IV rat metastatic model, Starkey et al. (21) had previously shown that the
metastasis enhancing moieties in serum from tumor-bearing rats reside with the IgG2b fraction. Since
all our previous work was done using the lung colony assay, the current study verified that metastatic
(but not local) tumor enhancement could also be demonstrated in a spontaneous metastasis system.
However, the lung colony assay was clearly most advantageous for quantitation. Modulation of the
immune system by TBS (serum from tumor-bearing rats) during the enhancement of metastasis was
studied. It was found that the Helper:Suppressor T cell ratio (H: S ratio) for peripheral blood leukocytes
(PBLs) from enhanced tumor-bearing rats was greatly reduced when compared to nonenhanced
tumor-bearing rats. The kinetics of this decline were also investigated. The H:S ratio for enhanced
tumor-bearer splenoc ytes and PBLs started to decline as early as day four and continued to do so until
the end of the experiment on day twenty. On the other hand, the H : S ratio for nonenhanced
tumor-bearing rats transiently peaked on day four and declined thereafter. Flow cytometric analyses of
lymphocytes from non tumor-bearing rats and tumor-bearing rats revealed that IgG from TBS bound to
a subset of T lymphocytes. Similar results were obtained for lymphocytes removed from enhanced
tumor-bearing rats. IgG2b was the predominantly binding isotype for enhanced tumor-bearing PBLs
and it appeared to be bound in vivo; binding by other isotypes was not significant. Preferential binding
of one isotype by nonenhanced tumor-bearing and non tumor bearing PBLs was not demonstrated.
Tumor bearer splenocytes, sorted on the basis of their ability to bind TBS IgG, were shown to enhance
experimental metastasis in the lung colony assay. All of the sorted cells were T lymphocytes and a
majority of the T cells expressed the suppressor T cell phenotype. These studies suggest that suppressor
T cells are involved in humoral enhancement of metastasis. The presence of these suppressor cells may
also explain problems associated with the adoptive immunotherapy of cancer. HUMORAL ENHANCEMENT OF METASTASIS:
CIRCULATING IgG INTERACTIONS WITH
TUMOR-BEARING LYMPHOCYTES
by
Cheryl Juline Aslakson
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Microbiology
MONTANA STATE UNIVERSITY
Bozeman, Montana
November 1986
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COPYRIGHT
by
Cheryl Jul in e Aslakson
1986
All Rights Reserved
iii
APPROVAL
of a thesis submitted by
Cheryl Jul in e Asia ks on
This thesis has been read by each member of the thesis
committee and has been found to be satisfactory regarding
content, English usage, format, citations, bibliographic
style, and consistency, and is ready for submission to the
College of Graduate Studies.
M /Teo
Date
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Approved for the Major Department
Date
Head,Major Department
Approved for the College of Graduate Studies
Date
^ 2
Gr ad Uatev D ea n
^
iv
STATEMENT OF PERMISSION TO USE
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the
require men ts
University ,
available
quotations
this thesis
for
a master's
I agree
that
to borrowers
from
this
in partial
the
are
of
degree at Montana State
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under rules
thesis
fulfillment
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of the Library.
allowable without
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Brief
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Request for permission
reproduction
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thesis
for extended quotation from or
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granted by the copyright holder.
7 Z
or
in
parts
may
be
ACKNOWLEDGMENTS
I would especially like to a c k n o w l e d g e Dr. Jean
Starkey for the guidance and inspiration that was rendered
during the course of this thesis.
I would also like to
acknowledge Dr. Sandra Ewald and Dr. Clifford Bond for
their helpful discussions and guidance..
Lastly, I wish to
thank my family and my fellow graduate students for their
interest and support.
vi
DEDICATION
I would like to dedicate this thesis in memory of my
f a th er , John Conrad A s l a k s o n.
It is his interest in
education which has sustained and given me the courage to
complete my master's degree.
vi i
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS.......................................
v
DEDICATION.............................................
vi
LIST OF TABLES.................................
ix
LIST OF FIGURES................ ..... ....... ..........
x
ABSTRACT...............................................
xi
INTRODUCTION...... ...................... .............
I
MATERIALS AND METHODS.................................
13
Rats.................. ,..........................
Tissue Culture Media....... ................... .
Tumor Cell Lines........
Sera................ ........... ..............
Spontaneous Metastasis Assa y...................
Lung Colony Assay........... ....................
Determination of Leukocyte Surface Phenotype...
Lymphocyte Preparation.... ............... .
T Cell Determinants.... ...................
B Cell Determinants. . . .....................
> Fluorescence Activated Cell Sorting Analyses...
Animal and Cell Preparation...............
Binding Parameters of IgG from NRS
and TBS............... ..............
Binding Parameters of IgM, IgGi, IgGza;
IgGgb and IgGgc from NRS and T B S ....
Fab Antibody Fragment Preparation.........
Adoptive Transfer of Splendcytes
Previously FACS-Sorted on the Basis
of Binding TBS IgG...................
Histological Examination........................
Statistical Analyses............... .............
RESULTS................................................
13
13
14
15
15
16
17
1,7
17
19
20
20
21
22
23
24
27
27
28
,
i
viii
Enhancement of Metastasis using the Lung
Colony Assay and the Spontaneous
Metastasis Assay.......................
Changes in I Cell and B Cell Phenotypes for
Leukocytes from Enhanced Tumor-Bearing
Rats and Nonenhanced Tumor-Bearing Rats...
Binding Parameters of Serum Immunoglobulin
to Lymphocytes ..............
Modulation of Tumor Lung Colonization with
Adoptively Transferred FACS-Sorted Tumor
Bearer Splenocytes...............
28
30
36
44
DISCUSSION.......................
49
REFERENCES CITED............
62
LIST OF TABLES
Table
1.
2.
3.
4.
3.
6.
7.
Page
The Effects of Tumor Bearer Serum on
Metastasis Using the Spontaneous
Metastasis Assay and the Lung Colony Assay....
29
Helper:Suppressor T Cell Ratios for "SerumEnhanced" Tumor-Bearing Rats, Nonenhanced
Tumor-Bearing Rats and Nontumor-Bearing Rats...
33
T and B Lymphocyte Counts for "Serum-Enhanced"
Tumor-Bearing Rats, Nonenhanced Tumor-Bearing
Rats and Nontumor-Bearing Rats........... * .....
37
Binding of Serum Immunoglobulin from TumorBearing Rats with Lymphocyte Populations from
Tumor-Bearing Rats and Nontumor-Bearing Rats...
39
Interactions of Serum IgG from Tumor-Bearing
Rats with Lymphocyte Populations from "SerumEnhanced" Tumor-Bearing Rats, Nonenhanced
Tumor-Bearing Rats and Nontumgr-Bearing Rats...
41
Immunoglobulin Isotype Binding Parameters for
Peripheral Blood Leukocytes from "SerumEnhanced" Tumor-Bearing Rats, Nonenhanced
Tumor-Bearing Rats and Nontumor-Bearing Rats...
43
Modulation of Tumor Lung Colonization by the
Adoptive Transfer of FACS-Sorted Tumor Bearer
Splenocytes........... ...........................
45
X
LIST OF FIGURES
Figure
1.
2.
3.
The Effects of Chronic Injection of
Tumor Bearer Serum on Subcutaneous Tumor
Growth ..............................................
Page
31
Helper!Suppressor T Cell Ratios for "SerumEnhanced" Tumor-Bearing Rats and Nonenhanced
Tumor-Bearing Rats...........................
Section of Lung Tissue from a Rat Injected
with 4bs Cells I .V . and FACS-Sorted
Splenocytes I. P .............................. .. .
35
46
xi
ABSTRACT
Using an inbred BD-IV rat metastatic model, Starkey et
al. (2 1 ) had previously shown that the metastasis enhancing
moieties in serum from tumor-bearing rats reside with the
IgGgb fraction. Since all our previous work was done using
the lung colony assay, the current study verified that
metastatic (but not local) tumor enhancement could also be
demonstrated in a spontaneous metastasis system. However,
the lung colony assay was clearly most advantageous for
quantitation.
Modulation of the immune system by TBS
(serum from tumor-bearing rats) during the enhancement of
m e t a s t a s i s was s t u d i e d .
It w a s f o u n d
t h a t th e
H e l p e r :Suppressor T cell ratio (H:S ratio) for peripheral
blood leukocytes (PBLs) from enhanced tumor-bearing rats
was greatly reduced when compared to •nonenhanced tumorbearing rats.
The kinetics of this decline were also
investigated.
The H :S ratio for enhanced tumor-bearer
splenoc ytes and PBLs started to decline as early as day
four and continued to do so until the end of the experiment
on day twenty.
On the other hand, the H :S ratio for
nonenhanced tumor-bearing rats transiently peaked on day
four and declined thereafter.
Flow cytometric analyses of
lymphocytes from non tumor-bearing rats and tumor-bearing
rats revealed that IgG from TBS bound to a subset of T
lymphocytes.
Similar results were obtained for lymphocytes
removed from enhanced tumor-bearing rats.
IgGgb was the
predominantly binding isotype for enhanced tumor-bearing
PBLs and it appeared to be bound in vivo; binding by other
i so types was not significant.
Preferential binding of one
isotype by nonenhanced tumor-bearing and non tumor bearing
PBLs was not demonstrated .
Tumor bearer splenocytes,
sorted on the basis of their ability to bind TBS IgG, were
shown to enhance experimental metastasis in the lung colony
assay.
All of the sorted cells were T lymphocytes and a
majority of the T cells expressed the suppressor T cell
phenotype.
These studies suggest that suppressor T cells
are involved in humoral enhancement of metastasis.
The
p r e s e n c e of t h e s e s u p p r e s s o r cells may also explain
problems associated with the adoptive immunotherapy of
cancer.
I
INTRODUCTION
The
demonstration
methyIchoI a n t h r e n e - induced
mice,
was
the foundation
by
Foley
sarcomas
of tumor
in
were
1953
that
immunogenic
immunology
(for
in
review
see Brodt in reference I, Hellstrom et al. in reference 2).
Foley
removed
mouse
was
established
resistant
Corroborative
(3),
an
reports
and in 1960,
These
studies
to
sarcoma and showed
a
second
followed
in
by Old et al.
excluded
tum o r
1957,
that
challenge.
by Prehn and Main
(4) and Klein et al.
the
the
possibility
antigenicity of methylcholanthrene-induced
(5).
that
tumors
the
was the
result of genetic heterogeneity in the inbred mouse strains
used
(3,4).
autochthonous
Klein
host
et
al.'s
could
also
data
revealed
be immunized
to
that
some
the
extent
against challenge with cells of its own tumor (5).
Tumor immunogenicity is not a phenomenon restricted to
chemic ally-induced
melanomas
are
reference
tumors.
also
6 ).
Ultraviolet
immunogenic
(for
Surprisingly,
(UV) light-induced
review
many
see
human
Kripke
tumo rs
in
and
spontaneously arising tumors in experimental animals proved
to
be
either
weakly
immunogenic
or non-immunogenic
(for
review see Brodt in reference I, Baldwin in reference 7).
The
tumors
immunity
invoke
that
c h e mic ally-induced
is cell-mediated
and has
experimental
been transferred
2
by
immune
cells
from tumor-bearing
hosts
(for review
see Hellstrom et al. in reference 2, North in reference 8 ).
Further
were
ijn vitro studies revealed
thymus-derived
reference
8 ).
lymphocytes
Passive
that the
immune cells
(for review
immunization,
the
see
North
practice
in
of
injecting tumor-bearing animals with pooled syngeneic tumor
bearer serum, did not confer immunity (for review see North
in reference 8 ).
Six
years
reformulated
the
discovery,
Burnett
Paul Erlich's pre-immunology theory
regarding
relative
after
lack
of
Foley's
tumors
in
animals
and
humans
(for
review see Brodt in reference I, Schwartz in reference 9).
This theory,
assumed
an
referred to as the immune surveillance theory,
active
role
for
thymus-dependent
cellular
mechanisms in searching out and eliminating cancerous cells
in situ. The
appearance
on
of
the
part
function
(for
the
host's
review
theory was later
of any tumors indicated a failure
see
immune
system to
Brodtin
reference
macrophages
cellular
I).
this
This
expanded and revised to include the non-T
cell dependent cytotoxic cells such as natural
cells,
fulfill
and cells mediating
cytotoxicity
killer
(NK)
antibody-dependent
(ADCC)
(for
review
see
Baldwin
have
been
raised
against
in
reference 1 0 ).
Many
objections
immune
surveillance as the means for controlling the appearance of
3
cancerous
animals
cells
(9,11,12,13).
and human patients
lymphoreticular
surveillance
types.
with
of
system
theory,
First,
develop
tumors
tumors
deficiency
(12,14).
tumors
(11,12,13).
should
S e co nd, nude mice which
immune
i mm unosuppressed
of
the
According
to
the
arise
in
all
are athymic and
syndromes
It has
mainly
do
been
not develop
argued
that
tissue
patients
an excess
the
lack
of
thymus-dependent cellular mechanisms is compensated for by
natural surveillance mediated by NK cells, macrophages and
cells mediating ADCC.
levels
NK
and
cells
develop
may
not
Athymic nude mice have high NK cell
very few
be
the
spontaneous
onl y
abrogation of ijn situ tumors.
cells
tumors
(12*13).
responsible
for
Beige mice which have low NK
levels develop spontaneous tumors at the same rate as their
normal
littermates
H o w e v e r , the
and
complexity
nud e
of
the
crystallize within the confines
theory.
At the
mouse
strains
cancer
problem
of the immune
time the theory was proposed,
answers to perplexing
questions
(12,13).
did
not
surveillance
it afforded
and spurred much research,
opening up new areas of study in tumor immunology.
The demonstration
followed
the
by
attempts
surveillance
active
immunity
that
tumors ,were
to explain
how these
mechanisms of their
to tumors was
immunogenic
hosts.
mediated
was
tumors escaped
Even
though
by T lymphocytes,
studies then focused on why injections of serum from tumor­
4
bearing
animals
prolonged
into
tu m o r
enhancement,
tum o r
survival
th e
a tumor and
its
using
tumor
tumor
bearer
resulted
in
Immunologic
to
describe
this
the successful establishment of
growth
resulting in the death
Tumor growth has been passively enhanced
bearer
serum
alloantibod ies
idiotypic
adopted
progressive
of the host (18).
hosts
(15,16,17).
phrase
phenomenon, was defined as
bearer
serum
(2 1 ,2 2 ),
(17),
antibody
antibodies
(25,26,27),
immune
fractions
antilymph ocyte
monoclonal
antibodies
(28,29,30,31),
(19,20),
factors
(23),
(24),
anti-
complexes
and
F (a b 1 )2
immunoglobulin
fragments
is not limited
to primary tumor growth; metastatic disease
has also been enhanced
Almost
all
(22,33).
serum
immune
(32)
from
Immunologic enhancement
(21,24).
antibody
classes have been
implicated
at
one time or another as being able to enhance tumor growth.
In the
rat
fraction
IAR6-l-RT7-4b
of
tumor-bearing
metastasis (21).
sarcoma
Sa I in
classes
IgG2 and
the
mineral
hepatocarcinoma model,
oil
serum
enhanced
the IgG^b
exp e r i m e n t a l
Allogeneic enhancement of the A/J-derived
CBA mice was attributed
IgGj
by Due et al.
plasmacytomas,
to
(15).
M0PC-315
the
The
and
antibody
growth of
M0PC-460,
induced in BALB/c mice was enhanced by the IgGj fraction of
alloantibodies prepared in CBA or CEH mice (17).
al.
(24)
prepared
monoclonal
Sacchi et
antibodies to a Lewis Lung
5
Carcinoma-associated
antibody
and
135-13C
antigen and
affected
its metastases
the
found that the monoclonal
growth of the
differently.
primary tumor
The primary
tumor
showed
a reduction of 20-25% in tumor weight compared to controls,
while
lun g
metastases
we re
increased
Antibodies of the IgG subclass
ability
to
enhance
tumor
are
growth.
(24) .
not exclusive;in
IgM
occasionally been implicated in prolonging
tumor tissue
two-fold
their
antibodies
have
the survival
of
(17).
Investigators have also reported that similar antibody
subclasses
against
are
also
effective
experimental
cancers
imm uno thera peuti c
(34,35,36).
agents
Denkers
et al.
(34) have shown that significant inhibition of the AKR/J SL
2 lymphoma
antibody.
cells
IgG%,
activity (34).
inhibit
was
Herlyn and Koprowski
of a human
Ig G2 a a n t i b o d y .
plasmacytoma
by the anti-Thy-1.1 IgG2a
IgG2b and IgG2c displayed less anti-tumor
the growth
monoclonal
mediated
MOPC
315
by
the
(35) were also able to
tumor in nude mice
Suppression
of
using a
the
mouse
IgG2 fraction of syngeneic
antitumor globulin also corroborates the fact that the same
isotypes which
enhance
tumor growth
in one system are the
tumor rejecting i so types in another system (36).
Differences seen in the ability of a given isotype to
enhance or restrict tumor growth may be attributed to many
factors.
These include the route of serum administration,
6
the
dilution
factor of the serum
and the methods
used
to
prepare the i so types or serum for injection.
Many
mechanisms
have
been
enhancement of tumor growth.
of
anti-tumor
complexes
from
or
antibody,
These
cells
and/or
blocking
protecting
them
Alternatively,
ineffective
(30)
were
bearing
circulating
animals
from
these
by binding
able
serum
to
by
found
lymphocytes
could conceal
immune
to them
remove
absorbing
in
serum
of binding
to
on tumors,
cells
(37,39).
the
the
(30,37,38,39).
render
blocking
in the form
antigens
effector
factors could
explain
ant ige n-antibody
may be capable
effector
factors
to
Blocking factors
free-circulating antigen
tumor-b ear ing
target
proposed
(37,39).
effector cells
Oldstone
factors
serum with
et
from
aI .
tumor-
the appropriate
tumor cells.
Specific classes of immunoglobulins in rodents and man
are
capable
of
which
activate
Ig G 2
and
classes
activating
the
IgG3
complement.
human complement
(40).
Analogous
in the mouse are IgM,
Immunoglobulins
system are
complement
IgGi,
activating
IgGgb
(41) and in
the rat are IgGj, IgGga , IgGgi3 and IgGgc (42).
Complement
activation
and
target
cell
IgGga and
IgM,
lysis
are
important
defense
mechanisms against tumor cells (8 ,10 ).
Antibodies
instead
enhance
which
tumor
do
not
growth.
activate
complement
Bodurtha
et al.
may
(43)
7
reported
on
antibodies
in
the
presence
of
ten
patients
with
relation to tumor metastasis.
patient
wi t h
extensive
complement-dependent
metastatic
possessed
com ple men t-d ep en den t
(43).
no
melanoma
disease
cytotoxic antibodies.
who
sera
malignant
in
They found that one melanoma
patients,
their
had
complem en t-activating
evidence
of
The other nine
visceral
cytotoxic
Demonstration
of
lacked
metastases,
antibodies
anti-com ple ment
in
(AC)
activity which may be due to anti-complement antibodies has
been documented
activity
in
humans
sometimes
reported
by
demonstrated
malignant
coincident
Gupta
in 45%
with
et
aI .
of the
with
(44).
sera
melanoma.
AC
tumor recurrence was
AC
activity
drawn from human
was
patients
with malignant melanoma compared to 10% of normal, healthy
controls
(44) .
Complex
host's
(45)
immune
interactions
response
formulated
described the
linked
and
together
via
immunoglobulin
V-region
where
idiotypes.
one
between
it.
In 1972,
a complex
complem entar y
mediated
a tumor
and its
Niels
Jerne
the network hypothesis which
immune system as
are
as
against
proposed
interactions
to
exist
by
network of cells
interactions.
structures
These
encoded
in
genes and have since been referred
The immune
system
exists
as
a network
idiotype is balanced by an anti-idiotype, which
in turn is counter-balanced
by an anti-anti-idiotype,
and
8
so
forth.
Via
maintained
perturbed
in
this
a complex
by
the
review
see Monroe
host's
control
antigenic
in reference
over
epitope
induce
state
of
immune
the
a foreign
46).
body's
system
homeos tas is
toward
agent
a
response
is
until
Alterations
responsiv ene ss
affects
(for
in
the
specific
to
further
Eichman et al. demonstrated that anti-
antibodies
can
specif ica lly
suppress
(47)
or
(48) immune responses to a particular antigen.
Idiotype
networks
have
also
immune control of tumor growth.
(49),
the
of
int roduction
antigenic stimuli.
idiotypic
network,
enhanced
tumor
growth
idiotypic tumor antibodies.
been
implicated
the
In a study by Flood et al.
was
attributed
to
anti-
These investigators found that
a UV-induced melanoma was able
to
result
suppression
of
in
id iot yp ic- spe cific
grow progressively
of
the
as a
tumor-
specific immune response to the melanoma antigens
(49).
corroborative
(25,26)
reported
and anti-idiotypic
cellular
study by Milburn
on the anti-idiotypic antibody
regulation
of
plasmacytoma.
and Lynch
IgA
expression
in
The
ant i-id io typ ic
the
MOPC-315
antibody
A
B A L B/c
regulated
expression of the immunoglobulin while the anti-idiotypic T
cells
regulated
other
hand,
secretion of immunoglobulin
inhibition
induced by anti-idiotypic
immunoglobulin idiotypes
of i_n vivo tumor
(25).
growth
antibodies directed at
on the surface of
the
On the
has been
distinct
murine
9
plasm acytoma
antibody
W0PC-460
8 .2 , an
(50) and
antibody
against
specific
murine
monoclonal
for a human
melanoma-
associated cell surface marker P 97 (51).
It
has
been
confirmed
tumor-bearing
animals
can
and metastatic
disease.
many
times
passively
that
serum
enhance tumor
The mechanisms
involved
phenomenon have not yet been clearly defined.
from
growth
in
this
In addition,
it is well documented that the adoptive transfer of tumorassociated lymphocytes
growth.
T r ev es
lymphocytes
host
the
aI .
(52)
from tumor-bearing
resistance
(3LL).
et
(52,53,54,55) can also enhance tumor
to
the
fou n d
th at
enhancing
T
C57BL/6 mice suppressed the
syngeneic
Lewis
Lun g-C arc ino ma
T lymphocytes obtained from the enlarged spleen of
tumor-bearing
in vitro.
mice
were
cytotoxic
to 3LL target cells
However, these same spleen cells enhanced tumor
growth ijn vivo when mice were injected i.v.
of spleen
cells and tumor cells
Uniel
Trainin
and
al.
Using
that
thymocytes
stimulate
effect
was
(52).
(53) corroborate
or
3LL tumor
thymus-derived
growth
manifested
Results reported by
the
the same syngeneic tumor
data
line,
of
3LL,
Treves
et
they found
lymphocytes
in recipients.
by a higher
with a mixture
cou ld
This enhancing
number of tumor
takes,
acceleration of tumor growth and an increase in metastasis
(53).
10
The progressive growth of tumors has been
modulated by suppressor
T cells
by Cheng et a l . (57)
found
effector
mice
T cells
down-regulated
cells.
in
by
(10,56).
that
and
with
North
Results reported
the generation
growing
cy clo phosp ham ide
Berendt
shown to be
(58)
of immune
plasmacytomas was
sensitive
have
suppressor
provided
evidence
consistent with the hypothesis that concomitant immunity to
the
Me t h
A
generation
Fibrosarcoma
of
a
decays
mechanism
immunosuppression .
In
as
of
add i t i o n ,
a
result
of
the
T-cell-mediated
North
and
Dye
(59)
reported that the Ly l+2~ suppressor T cells down-regulated
the
generation
of
Ly
l- 2+ effector
T cells
progressive growth of a P815 mastocytoma.
findings
suggest
that the
failure
during
the
These and other
of the immune
system
to
reject an immunogenic tumor is the result of the generation
of suppressor T cells (54-59).
Suppressor
have
been
(60,61).
T cells derived
shown
to
suppress
from
immune
tumor-bearing
functions
hosts
iji vitro
Clerici et al . (62) reported that suppressor cell
activity,
enriched
tumor-bearing
by
mice,
hyd rocortisone
treatme nt
down-regulated
3 LL
i_n
vitro
bearer mouse
spleen
lymphoproliferative
response
cells to mitogens.
The study undertaken by Bear (63) found
that
spleen
cells
from
of tumor
an
of
"late"
tumor-bearing
hosts
(18-28
days post tumor cell inoculation) completely Suppressed the
11
in
vitro
cells.
cytotoxic
induced
allogeneic
depressed
counts,
response
to
tumor
immune
Mixed-lymphocyte culture responses of spleen cells
from tumors
against
lymphocyte
by the murine
spleen cells
as indicated
^H-thymidine
by
Moloney
were
Sarcoma
found
to be markedly
three parameters:
incorporation
Virus
lymphoblast
and cell-mediated lysis
(63).
Suppression of the immune system has been reported for
many
other
changes
this
diseases
and
has most
often been
in helper: suppress or I cells
suppressed
state are
patients
reflected
by
Included
in
ratios.
with
Acquired
Immune
Deficiency Syndrome (64), Systemic Lupus Erythematosus
and
leprosy
ratios
(66,67).
have not
Changes
in helper:suppressor
been widely reported
in
canine
lymphosarcoma.
T cell
for cancer patients.
Koya ma 'et a l . (68 ) did find changes in B and
counts
(65)
Marked
I lymphocyte
increases
in B
lymphocytes and decreases in I lymphocytes were observed in
the peripheral blood,
spleen and lymph nodes of dogs which
had been diagnosed as having lymphosarcoma (6 8 ).
Immune regulation
humoral
tumor
and
cellular
of tumor
growth is complex;
arms
the
proliferation.
alternatively, allowed
system.
of
The
to
tumor
grow
I chose to investigate
immune
may
both the
system
be
unchecked
one of the
control
destroyed
by
the
or
immune
manifestations
12
of unchecked
tumor
tumor metastasis.
growth,
the
immunologic
enhancement
of
13
MATERIALS AND METHODS
Rats
BD-IV
rats,
originally
obtained
RajeiAisky, Institute for Cell Biology,
Germany
and
Research
from
Dr.
on Cancer, Lyons,
conditions
France, were bred
Resources
at the Montana
Center.
P rof es sor
University of Essen,
Montesano, International
sister matings and maintained under
(SPF)
from
Agency for
by
brother-
specific pathogen free
State
University
Animal
six t o twelve weeks of age
Male rats,
were used in these studies.
Tissue Culture Media
The
R T7-4bs
tumor
RPMI
1640
medium
with
10%
fetal
Logan,
U/ml
line
(GIBCO, Grand
bovine
serum
was cultured
Island,
NY)
i_n vitro in
supplemented
(FBS) (HYCL ON E LABORATORIES,
U T ), 5ug/ml insulin (CALBIOCHEM, La Jolla,
penicillin
streptomycin
complete
and
cell
medium
RPMI
Mg+ ^
used
Grand
(GIBCO) .
intravenous
Ca+^ and
(G IB C 0,
for
medium.
This
Tumor
injections
-free
Island,
NY)
formulation
cells
were
used for
CA),
and
lOOug/ml
constitutes
subcutaneous
washed and resuspended
Tyrode's balanced saline
fluorescence-activated
100
(C M F ).
cell sorter
in
The
(FACS)
analysis of lymphocyte preparations and for FACS sorting of
splenocytes was McCoy's 5A medium (GIBCO) supplemented with
14
5 ?o heat- in ac ti va ted
buffered saline
FBS
with
(BOEHRINGER MANNHEIM
BSA) and
St.
D u l b e c c o 's
phosphate
0.5% bovine serum albumin,
fraction V
BIOCHEMICALS,
PBS-BSA with
Louis,
(CM).
0.01M
NaN5
Indianapolis,
I N )(PBS-
(SIGMA CHEMICAL
COMPANY,
MO) were used for the determination
of T and B
cell surface antigens.
Tumor Cell Lines
The tumor cell lines,
Ls),
were
derived
from
RT7-4bs (4bs) and RT7-4b-Ls (4b-
IAR6-1-RT7,
a dimethylnitrosamine-
transformed culture of BD-IV rat parenchymal cells (69,70).
Characteri zat ion
of
these
cells
lines
is
described
in
detail elsewhere (71,72). These tumor lines were maintained
in BD-IV
rats
by
subcutaneous
every three weeks.
cultures
through
from
a 200
RPMI medium.
twice
of
each
tumor
RT7-4bs cells were obtained as primary
minced
mesh
Cell
in complete
RPMI medium.
transplant
subcutaneous
stainless
tumor
steel
suspensions
screen
were
RPMI medium and
tissue
into
passed
complete
pelletized,
resuspended
washed
in complete
Cells were plated at I x 10^/sq cm in 25 cm^
tissue culture flasks (NUNC, Denmark) and incubated at 37°C
in a humidified atmosphere of 7% CO2 in air.
cell
lines
were
trypsinization
harvested
[0. 05 %
trypsin
ethylenediaminetetraacetic,
CHEMICAL
COMPANY)
in
for
CMF]
passage
by
(CALBI0CHEM)
d i sodium
for
Once a week,
salt
gentle
and
(EDTA)
five minutes
0.02%
(SIGMA
at
room
15
temperature,
RPMI
followed by trypsin inactivation with complete
medium.
The cells were
then replated at a 1:4 split
ratio.
Sera
Blood
under
was
ether
analogous
obtained
anesthesia
via
cardiac
bearing rats which two weeks earlier
had been
pieces
was
of
the
subcutaneously.
The
centrifuged
4 ° C , 7 50 xg
fractions
at
from
and pooled.
4b-Ls
tumor
Pooled
NRS
frozen
at
blood was
4bs
and
for
twenty
(TBS)
were
TBS were divided
- 2 0° C
an
tumor­
transplanted
tumors
minutes.
were
then
Serum
collected
the serum fractions from the 4bs and
bearing rats
and
from
4b-Ls
(NRS)
rats
In
allowed to clot , and
nontumor-bearing rats
Likewise,
male
puncture.
obtained
small
blood
nontumor-bearing
also
with
fashion,
from
until
use.
collected
into
and pooled.
aliquots
Freeze-thaw
and
stored
cycles
were
avoided.
Spontaneous Metastasis Assay
4bs tumor
cells
were
removed
from the tissue culture
vessels by gentle trypsinization as described earlier.
monodispers ed
with
CMF
cells/ml
and
tumor
cells
resuspended
in CMF .
Tumor
were
at
cell
pelletized,
washed
a concentration
viability
trypan blue dye exclusion and cell
was
The
twice
of 5 x 10&
assessed
suspensions which
by
were
16
greater
hundred
into
than 9 5 % viable were
thousand tumor
the right
for inoculation.
cells were
injected
flank of each rat.
cell injection,
Two
each rat in the control
intra pe ri to neall y (i.p.)
the
used
experimental
with
group was
0.2ml
injected
Five
subcutaneously
hours
post
tumor
group was injected
NRS and
i.p.
each
with
rat
0.2ml
in
TBS.
Sera injections were continued every second day thereafter.
Subcu taneous
tumor
growth
was
monitored every
second day
with vernier calipers and tumor volume was calculated using
the formula 4/3 tt r^ .
At the time of necropsy, total body
weight was recorded and the rats were examined for evidence
of
metastatic
subcutaneou s
disease.
tumor
B o u i n 's fixative
Lungs,
were
(73).
liver,
removed,
The
spleen
weighed
number
of
and
and
the
fixed
spontaneous
in
lung
metastases was enumerated using a dissecting microscope.
Lung Colony Assay
RT 7- 4bs
tumor
cells
were
harvested
from the
tissue
culture vessels as described earlier and resuspended at 3 x
IO^ cells/ml in CMF.
previously
inoculum
Tumor cell viability was assessed as
described.
were
injected
rat.
Two hours
tumor
cells
and
after
every
Ten thousand
into
the lateral
intravenous
two
cells
days
(i.v.)
in
0.2ml
total
tail vein of each
injection of 4bs
thereafter,
rats in the
control group and the experimental group were injected with
serum as
described for the spontaneous
metastasis
assay.
17
Post-mortem
pulmonary
investigation
metastases
and
liver and spleen weights.
fixed
in
Bouin's
included
examination
recording
for
of total
extra-
body,
lung,
The lungs, liver and spleen were
fixative,
and the
number
of tumor
lung
colonies enumerated using a dissecting microscope.
Determination o f Leukocyte Surface Phenotypes
Lymphocyte
dispersed
Preparation.
Spleens
to single cell suspensions
200 mesh
wire screen.
Peripheral
were
removed
by passing
blood
and
through a
leukocytes
(PBLs)
were isolated from freshly drawn blood obtained via cardiac
puncture.
The blood was mixed with heparin (SIGMA CHEMICAL
COMPANY)
at
(50mg/ml;
4 0C , 750xg
removed.
0.1ml/3ml
for
twenty
of whole
minutes
blood),
and
the
Erythrocytes were removed from cell
by hypotonic
d is ti ll ed
water
resuspended
cells/ml.
lysis
in
using a six second
(74).
PBS-BSA
Cel l s
at
were
centrifuged
buffy
coat
preparations
exposure
to sterile
washed
twice
a concentration
of
and
2 x IO^
Two million cells per well were dispensed into a
flat-bottomed,
centrifuged
96-well,
at 4°C,
IBBxg
microtiter
plate
for five minutes and
(NUNC),
the excess
supernatant was removed.
T_ Cell
antibodies
SCIENCE,
Determinants.
The
following mouse
monoclonal
(mab), which were purchased from BIOPRODUCTS FOR
Inc.,
Indianapolis,
IN,
were
used
surface determinants on rat lymphocytes:
to
label
cell
18
1. )
W3/25.
This
subset
functional
graft
versus
mab
in
host
labels the rat helper
mixed
reactions
lymphocyte
T cell
reactions,
and antibody responses
(75,76),
2. )
MRC OX-8 .
mediates
also
This mab
suppression
labels a T cell subset which
of
antibody formation (77). It
recognizes a determinant expressed on
precursor
cytotoxic T cells (78), and
3. )
MRC OX-19.
This
mab
recognizes
a
determinant
expressed on all thymocytes and peripheral T cells but
does not
bind to B cells, macrophages, natural killer
cells, mast cells or other cell types (78).
The
indirect
technique
was
immunofluorescence
used
to
determine
(HF )
microtitration
T lymphocyte
subsets.
Fifty ul of a 1:50 dilution of mabs W3/25, MRC OX-8 and MRC
OX-19 was
4°C
for
added to
one
hour.
PBS-BSA
containing
second
antibody,
conjugated
the centrifuged
cells
After
were
the cells
azide,
a
washed
i s o t h i o c y n ate
IgG antibody
at
twice with
50uI of a 1:32 dilution
fluorescein
goat anti-mouse
and incubated
of the
(FITC)
(CALBIOCHEM)
was
added and the incubation continued for another hour at 4°C.
The cells were washed twice in PBS-BSA containing azide and
resuspended in PBS-BSA containing azide
(79).
cells
preparation
from
each
labeled
cell
One hundred
were
19
counted
and
evaluated
Ortholux II microscope
The
for
fluore sce nce
using
a Leitz
(E . LEITZ, Inc., Rockleigh,
he lpe r:s upp res sor
T cell
ratio
(H:S
NJ).
ratio)
was
expressed as follows:
# positive fluorescent cells labeled by IaJ3/25
#
positive fluorescent cells labeled by MRC OX-8
The percentage of T cells was expressed as the:
# positive fluorescent cells labeled by MRC OX-19
x 100.
# total cells counted
B_ Cell
procedure,
(80),
Determinants.
The
I IF
antibody
labeling
a modification of the method described by Moller
was
milliliter
used
to
of a 1:64
label
B
lymphocytes.
dilution
of
a rabbit
antibody (MILES LABORATORIES, Elkhart,
One-tenth
anti-rat IgM
IN) was added to the
centrifuged cells and incubated at 4°C for one hour.
After
the cells were washed twice with PBS-BSA containing
azide,
0.1ml
of
conjugated
Inc.,
1:100
goat
Malvern,
dilution
of the
second antibody,
anti-rabbit antibody
PA)
was added
and
for an additional hour at 4°C.
(COOPER
a FITC-
BIOMEDICAL,
the incubation
continued
The cells were washed and
resuspended in PBS-BSA containing azide.
One hundred cells
were counted using a fluorescence microscope
and evaluated
2D
for fluorescence.
The percentage of B cells was expressed
as :
# positive fluorescent cells labeled by anti-IgM
x 100.
# total cells counted
Fluorescence Activated Cell Sorting Analyses
Animal and Cell
Preparation.
Male rats were prepared
for FACS analyses in an analogous fashion for those used in
the experimental lung colony assay.
Ten thousand 4bs tumor
cells were injected into the lateral tail vein of each rat.
Two hours
after the tumor cells were injected, each rat in
the control group was injected with 0.2ml NRS i.p. and each
rat
in
the
experimental
injections
were
fourteen.
On day
and
experimental
bearing
group with 0.2ml
continued
every
fourteen,
the
groups and two
rats were
bled under
was
described,
centrifuged
minutes.
Spleens
Serum was
and
were
from
heparin
at
A 0 C,
and
the
removed
as
7 50 xg
buffy
and were
Sera
unt i l
day
the control
age/sex matched
with
discarded
thymuses
animals
day
ether anesthesia
p u n c t u r e . Blood
and
mixed
second
TBS i.p.
nontumor­
via cardiac
previously
for
twenty
coat
saved.
dispersed
to
single cell suspensions by passing through a 200 or 70 wire
mesh screen,
cell
respectively.
preparations
distilled
water as
using
Erythrocytes were removed from
hypotonic
lysis
described earlier.
with
The
sterile
nylon
wool
21
nonadheren t
incubation
five
splenocyte
were
collected
of splenocytes on nylon wool columns
minutes
washed
fractions
at
twice
37°C (81).
with
CM
and
All
after
for forty-
cell suspensions were
resuspended
in
CM
at 2 x IO^
cells/ml.
Binding
Preliminary
Parameters
experiments
o_f
IqG
revealed
from
that
NRS
and
the optimal
IBS ,
working
dilutions for NRS, TBS and the FITC-conjugated rabbit antirat
IgG antibody
were
1:1,
1:12.5
and 1:50,
respectively.
One-tenth milliliter of each cell
suspension was incubated
with
minutes
CM,
NRS
or
TBS
for
thirty
at
37°C.
After
washing twice with CM, the cell suspensions were incubated
with
0.1ml
(MILES
FITC-con jug ated
YEDA, Ltd,
LABORATORIES)
Rehovot, Israel,
for thirty
washed twice with
rabbit
anti-rat
IgG
antibody
distributed
minutes at 370C.
by MILES
The cells were
CM and resuspended in a total
volume
of
1.0ml CM.
The
intensity
cells was
of
determined
fluorescence
using
of the TBS
IgG-Iabeled
a fluorescence-activated
cell
sorter, FACS 440 (BECTON-DICKINSON, MOUNTAIN VIEW, CA). The
FACS 440 was
interfaced with
the
LACELL
programs,
The
488
line
nm
of
ACQ4
the
Consort
and DISP
a 164-02
40
equipped
with
4 (BE CT ON-DICK INS ON ).
Argon-ion
laser
( SPECTRA
PHYSICS, Mountain View, CA) operated at 100 mW, was used to
excite the
fluorochrome.
Forward
scatter,
90
degree
22
scatter and fluorescence intensity were acquired on samples
of 10,000 events using the ACQ4 program.
was
used
to
reflecting
calculate
the
amount
with NRS and TBS.
with
CM
was
the
The DISP 4 program
intensity
of rat
of
fluorescence,
IgG bound after
incubation
The data gathered from samples incubated
used
to
discriminate
between
nonspecific
fluorescence and fluorescence emitted from TBS IgG-Iabeled
cells.
and
Contaminating
debris
were
traces
excluded
of red cells, n enviable cells
by
appropriately
setting
the
I q G ya , I g G yh
and
forward scatter threshold (82).
Binding
Ig Gy n from
Parameters
NRS
and
o f I q M , I q G i,
TBS.
Commercial
antibodies
used for
these studies included the following:
1. )
The
FOR
SCIENCE,
monoclonal
monoclonal
antibody MARG 2b-8
Inc.).
IgGj antibody
This
antibody
prepared
(BIOPRODUCTS
is
mouse
against the gamma2b
heavy chain of the rat IgGyb immunoglobulin
2. )
a
(83), and
The polyclonal antibodies:
a. )
Goat anti-rat IgM (COOPER BIOMEDICAL,
b. )
Sheep anti-rat IgGj
c. )
Goat anti-rat
Rogers, AR),
d. )
Inc.),
(MILES LABORATORIES),
IgG2a (PEL-FREEZ BI0L0GICALS,
and
Goat anti-rat IgG2c (MILES LABORATORIES).
These polyclonal antibodies were conjugated to FITC (COOPER
23
BIOMEDICAL,
Hapner
using the method developed
by Hapner and
(84).
PBLs
antibody
37°C.
Inc.)
were
incubated
with
CM,
NRS, and TBS
(using the
dilutions given on page 21) for thirty minutes
Cell
preparations were incubated
further
with
at
MARG
2b-8 for thirty minutes at 37°C followed by incubation with
a FITC-conjugated
goat
more minutes at 370C .
anti-mouse
Similarly,
IgG antibody
for thirty
following incubation with
CM, NRS, and TBS, the same cell preparations were incubated
with the conventional F ITC-conjugated antibodies for thirty
minutes
at
3 7 ° C to
determine
the
binding
parameters
of
these antibody isotypes from NRS and TBS to RBLs.
The labeled cells were analyzed on the FACS 440 using
the
same laser
programs
positive
as
setting,
and
pr evi ous ly
fluorescent
data
acquisition and
described.
cells
was
The
analysis
percentage
calculated
as
of
previously
described under Binding Parameters of IgG from NRS and TBS.
Fab
of
TBS
Antibody Fragment Preparation.
was
ammonium
obtained
sulfate
enzymatically
BIOMEDICAL,
Methods
in
by
Inc.)
precipitation
(SI GM A
digested
CHEMICAL
wi t h
using the
Cellular
with
described
(85).
Fab
fraction
saturated
COMPANY)
material by
Protein A-Sepharose CL-4B column
IgG
and
mercuripapain
method
ImmunoIo qy
purified from the digested
The
in
then
(COOPER
Selected
fragments
passage over
were
a
(PHARMACIA FINE CHEMICALS,
24
Sweden)
and diluted to a final volume
equal
to the 1:12.5
TBS dilution used for other FACS analyses.
The quality of
the
a 1.5%
papain
digestion
was
assessed
using
Sodium
Dodecyl Sulfate-PAGE gel under nonreducing conditions (86).
PBLs
were
antibody
incubated
dilutions
fragments
as
with
given
CM,
on
previously
NRS
page
and
21)
described
Parameters of IgG from NRS and TBS.
TBS
and
for
(us in g
the
antibody
the
Binding
The labeled cells were
analyzed on the FACS 440 using the same laser setting,
data
acquisition
described.
and
Also, the
analysis
percentage
the
programs
as
of positive
and
previously
fluorescent
cells was calculated as previously described under Binding
Parameters of IgG from NRS and TBS.
Adoptive
Transfer
o f Splenocytes
Sorted on the Basis of Binding
tumor cells,
the
BD-IV
spleen
suspension
cells
was
by
insulin,
Two weeks
removed
passing
were
distilled
were
Ten thousand 4bs
were injected into the lateral
rat.
Erythrocytes
sterile
TBS IqG.
FACS-
prepared as previously described for the lung
colony assay,
a male
Previously
washed
lOOug/ml
and
through
as
a 200
using
cell
to
mesh
wire
cell
screen.
lysis
with
described.
CM supplemented
and lOOU/ml
injection,
a single
hypotonic
previously
twice with
penicillin
tumor
dispersed
removed
water
after
tail vein of
with
The
5ug/ml
streptomycin
resuspended at 2 x 10? cells/ml in supplemented CM.
and
Twenty
25
million
cells
antibody
37°C.
were
dilution
incubated
with
given on page
1.0ml
21)
TBS
(using
the
for thirty minutes
at
The cells were washed with supplemented CM and then
incubated with
1.0ml
antibody
thirty
for
F ITC-conjugated
minutes
at
rabbit
37°C.
anti-rat
The
cells
IgG
were
sorted, using the 488 nm line of the 164-02 Argon-ion laser
operated
Channel,
laser
at
I 00
mW in
mode
on
the
Fluorescence One
on the basis of binding TBS IgG.
and settings
analogous
patterns
log
to
those
for the
used
of antibodies
sorted over two hours,
forward
for
from
Operation of the
scatter
threshold were
asc ert ai ni ng
NRS
and TBS.
the
The
collected into complete
and held on ice until the sort was completed.
binding
cells
were
RPMI medium
Five hundred
thousand IgG positive-sorted cells were injected i.p.
into
each of five experimental rats, which two hours earlier had
been
injected
with
I x IO^ 4bs
described
for the lung
had
been
also
colony
injected
tumor cells
assay.
two hours
as
Control
previously
rats, which
earlier with
4bs tumor
cells i .v ., were injected i.p. with 0.2ml 0.85% NaCl (SIGMA
CHEMICAL COMPANY).
Three weeks after tumor cell injection,
the animals were killed and necropsied as described for the
lung colony
assay.
Tumor lung colonies
were counted under
a dissecting microscope.
I g G - p o s i t i v e , sorted
splenocytes
were
typed
splenocytes
by
and
11F for helper
unsorted
T
cells,
26
suppressor
T cells
splenocytes
1.0ml TBS
21)
for
were
and
total
prepared
for
I cells.
Twenty
sorting by incubating
(using the same antibody dilutions
thirty
million
minutes at 37°C.
with
given on page
After washing twice with
CM, the cells were incubated with 0.1ml of a 1:1 dilution a
rabbit
anti-rat
conjugated to
BIOMEDICAL,
IgG
antibody
(COOPER BIOMEDICAL,
Inc.)
rhodamine tetramethyl isothiocyanate (COOPER
Inc.)
as previously
described
for FI TC .
The
fluorescently-labeled splenocytes were sorted on the basis
of IgG bound
laser was
filter
from
TBS.
The 514 nm line of the Argon-ion
operated at 100 mW with the DF 575/25
in place,
and the sorting
was
set
which
been
sorted
had
as
pass
was monitored in the log
mode on the Fluorescence Two channel.
threshold
band
previously
The forward scatter
described
and injected
for
the cells
for adoptive transfer.
The cells were collected into complete RPMI medium and held
on ice as previously
hours
in
complete
described.
RPMI
medium,
After
the
incubation
for two
sorted splenocytes
as
well as unsorted splenocytes were labeled with mabs W3/25,
MRC OX-8 and MRC OX-19.
Labeled cells were counted and the
data calculated as previously described under Determination
of Leukocyte Surface Phenotypes.
When
sorted
it
was
discovered
splenocytes,
fluorescein,
would
that
labeled
not
TBS
with
complexes
either
cap off even
on
FACS
rhodamine
after
or
overnight
27
incubation
at
37°C,
similar
cel l
preparations
subjected to light trypsin treatment in
the complexes.
and
anti-rat
0,1ml trypsin
Following
attempts to remove
After the splenocytes were labeled with TBS
FITC-con jugated
described,
were
trypsin
to two minutes
minutes,
the
complete
R PM I .
was
addition,
and then
cells
IgG
antibody
added
recovered
Following
previously
to the splenocytes.
at thirty-second
at two minute
were
as
intervals
intervals
from
trypsinization,
up
up to ten
trypsin
with
the cells
were
assessed for complex removal using fluorescence microscopy.
Histological Examination
Lungs
and
subcutaneous
tumors
from
animals
in the
various experimental and control groups were excised, fixed
in
Bouin's
embedding.
solution,
Sections
and
processed
were
stained
routinely
with
for
paraffin
hematoxylin
and
eosin (H & E ) or Periodic-Acid Schiff and hematoxylin (PASH ).
Statistical Analyses
Differences
experimental
in the
number
of lung met as tase s between
groups were analyzed using the non-parametric
Mann-Whitney
U
Two-Tailed
significant at p < 0.02.
Test
and
were
considered
28
RESULTS
Enhancement
of Metastasis u sinq
the Lung Colony
Assa y and
the Spontaneous Metastasis Assay
Chronic injection of TBS enhanced lung colonization by
the 4bs tumor using the experimental lung colony assay.
Table
I sh o w s , chronic
colonization
NRS.
for
2.7
injections
of
times over control
TBS
As
enhanced
lung
injected
with
animals
The average number of pulmonary tumor nodules was 173
enhanced,
experimental
rats
and 63
for control
rats
(Table I.)
The
spontaneous
investigate
whether
spontaneous
lung
analogous
was
with
Values
TBS.
enhanced",
tumor
lung
subcutaneous
metastasis
fashion.
metastasis
metastasis
As
shown
enhanced
in
tumor lung nodules
Chronic
si gn ificant ly
subcutaneous
in
for spontaneous
and
from
be
growth
modulated
I,
to
and
in
an
spontaneous
metastases
control
used
chronically
correspond
injected
from
"serum-
to 38 metastatic
rats,
17
metastatic
(Table I).
injection
of
affect
growth
tumor
wa s
tum o r
Table
animals
experimental rats
nodules
could
ass ay
the
(Figure
I.)
TBS,
however,
rate
of
the
did
not
primary,
As shown in Figure I, the
latent period for the appearance of the primary tumors and
the growth rates were similar for both groups.
29
TABLE I.
THE EFFECTS OF TUMOR BEARER SERUM
ON METASTASIS
USING THE SPONTANEOUS METASTASIS
ASSAY AND THE LUNG
COLONY ASSAY
REGIMEN
RAT GROUP
MEAN NUMBER OF
PULMONARY TUMOR
COLONIES
(RANGE) pa
Controls
65 (35 - 120)
LUNG COLONY ASSAY
BD-IV rats were injected
with IxlO^ 4bs cells i.v.
Two hours after 4bs cell
injection, control rats
received 0.2ml NRS*3 i.p.
and experimental rats 0.2
ml TBS*3 i.p.
Sera
injections continued
every second day until
necropsy at Day 20.
(n
= 5)
Experimentals
(n = 5 )
173 (94 - 457)
P < 0.0087
Controls
(n = 8 )
17 (I - 63)
Experimentals
(n = 7)
58 (0 - 263)
P =0.3
SPONTANEOUS METASTASIS
ASSAY
BD-IV rats were injected
with 5 x 105 4bs cells
subcutaneously.
Sera
injections were given in
a fashion analogous to
the lung colony assay
until necropsy at Day
27,
a p - values were calculated using Mann-Whitney U TwoTailed Test.
^NRS = serum from normal rats; TBS = serum from tumor
bearing rats.
30
Histologic examination of lung sets
and experimental rats showed
indeed tumor colonies.
from control rats
that the nodules counted were
There was no discernible difference
in lung morphology between the two groups.
In summary, metastatic
enhanced
using
spontaneous
the
tumor
experimental
metastasis
lung
lung
assay.
colony
Humoral
essentially restricted to metastasis.
enhanced
colony
using
assay,
quantitation,
Changes in
Enh anced
either
which
assay,
is
colonization
we
assay
was
and the
enhancement
was
Since metastasis was
chose
inheren tly
to
use
a better
the
lung
assay
for
for the remainder of these studies.
Cell and B_ Cell Phenotypes for Leukocytes from
Tumor Bearing
Rats
and
Nonenhanced
Tumor-Bearinq
Rats
TBS
modulation
enhancement
of
the
of metastasis was
immune
system
studied.
First,
during
the
changes
in
the H :S ratios were investigated for enhanced tumor-bearing
rats.
As
shown
in
Table
2,
fourteen
days
after
i.v.
inoculation of 4bs tumor cells, the H :S ratio for PBLs from
enhanced
tu mor- bea rin g
rats
was
g r eat ly
compared
to nonenhanced tumor-bearing rats.
for PBLs
from enhanced tumor-bearing
and
from
(Table 2).
nonenhanced
This
reduced
The H :S ratio
rats was 0.57 +_ 0.03
tumor-bearing rats was
reduction was
when
restricted
to
1.49 + 0.21
circulating
PBLs and was not seen for splenocyte preparations.
The H :5
9000
8000
7000
6000
CU 5 0 0 0
4000
3000
2000
1000
Treatment Day
FIGURE I. THE EFFECTS OF CHRONIC INJECTION OF TUMOR BEARER SERUM ON SUBCUTANEOUS
TUMOR GROWTH. BD-IV rats were injected with 5 x IO^ 4bs cells subcutaneously.
Two hours after tumor cell injection, control rats (•--- •--- #) received
0.2
ml NRSa and experimental rats ( O--- O--- O ) 0.2 ml TBSa. Sera injections were
continued every second day until the experiment ended at day 27.
aNRS = serum from normal rats;
TBS = serum from tumor-bearing rats.
32
ratios
for
splenocyte
non enhanced
and
0.89
tumor-bearing
+_ 0.21,
established
preparations
for
rats
from
were
respectively
enhanced
similar,
(Table
2).
nontumor bearer control
and
0.84 + 0.16
The
H :S ratios
rats were
1.63 +
0.33 for splenocytes and 1.46 +_0.10 for PB Ls .(Table 2).
Since
a major
"serum-enhanced"
da ys
after
reduction
in
tumor-bearing
tumor
the
inoculation,
Animals
moribund
time of tumor
cell
injected with 4bs
twenty-one
days
PBLs
H :S ratio
rats was detected
helper:suppressor modulation was
from
the
the
injection
tumor
cells
fourteen
kinetics
monitored
of
the
every four days
until
i.v.
after injection
for
day
twenty.
usually
become
tumor
cells.
of
As shown in Figure 2, the immune systems of enhanced tumorb e ar in g
rats
started
injection.
The
preparations
from
H :S
to
change
ratios
shortly
for
"serum-enhanced"
after
splenocyte
tumor
and
tumor-bearing
PBL
animals
started to decline as early as day four, although this was
not as marked
The
for splenocytes
H :S ratios
sixteen
when
splenocytes
terminated.
for
th ey
PBLs
continued
stabilized.
continued
The
(Figure 2,
to
lowest
drop
ratio,
to
The
until
Panels
A and B ).
decline
until day
H :5
the
which
ratios
for
experiment was
was
seen
on
day
twenty, was 0.53 +_ 0.05 for splenocytes (Figure 2, Panel A)
and 0.52 +_ 0.06 for PBLs (Figure 2, Panel B ) .
33
TABLE 2.
HELPER : SUPPRESSOR
T CELL RATIOS
FOR
"SERUMENHANCED"
TUMOR - BEARING
RATS,
NONENHANCED
TUMOR­
BEARING RATS AND NONTUMOR-BEARING RATS
SPLENOCYTES
(HELPER:SUPPRESSOR
+ S .E . )
PERIPHERAL BLOOD
LEUKOCYTES
(HELP E R :SUP PRESS OR
+ S .E . )
NONTUMOR­
BEARING RATS
(n = 4 )
1.63 + 0.33
1.46 + 0.10
NONENHANCED
TUMOR-BEARING
RATS
(n = 3)
0.89 + 0.21
1.49 + 0.21
ENHANCED TUMOR
BEARING-RATS
(n = 4 )
0.84 + 0.16
0.57 + 0.03
Ten thousand 4bs tumor cells were injected i.v. into
the lateral tail veins of four male BD-IU rats.
Two hours
l a t e r , two rats we r e i n j e c t e d i.p. wi t h 0.2ml N R S a
(nonenhanced tumor-bearing rats) and the remaining two rats
were injected with 0.2ml T B S a (enhanced tumor-bearing
rats).
Sera injections continued every second day until
day fourteen, at which time the helper:suppressor T cell
ratios were evaluated by indirect immunofluorescence.
a NRS = serum
bear i n g rats.
from
normal rats;
TBS = serum
from tumor-
34
On
the
exhibited
possibly
immune
other
a slightly
reflecting
activity
experimenta l
splenocytes
rats
hand,
at
day
has
(87)
respectively.
we r e
After
been
early
from
1.5 4
day
on
2).
bea rer s
day
four,
stimulation
reported
(Figure
PB Ls taken
tumor
H :S ratio
a generalized
as
four
nonenhanced
increased
systems
and
the
in
of
several
H :S ratios
for
nonenhanced tumor-bearing
+_ 0.02
four,
and
the
1.53
H :S
+
0.07,
ratios
for
splenocytes and PBLs started to decline and continued to do
so until the experiment ended
At the end of the experiment,
(Figure 2,
Panels
A and B ).
the HsS ratio was 0.71 +_ 0.02
for splenocyte and PBL preparations (Figure 2, Panels A and
B ).
Control
rats were
values
1.45
for age/sex matched
0.07
nontumor-bearing
for splenocytes and 1.45 +_ 0.08 for
PBLs.
HsS ratios changed for enhanced and nonenhanced tumorbearing
rats
concomitant
shown
in
but
changes
Table
significantly
percentages
3,
chan ge
in overall
B and
when
of T cells
"serum-enhanced"
1%.
this
was
B and
T cell
was
splenocyte
accompanied
I cell
numbers
metastasis
for
not
numbers.
did
not
by
As
change
enhanced.
The
prepa ratio ns
from
tumor bearers were 50 j^l?o and PBLs 49 +_
"Serum-enhanced"
splenocytes
cells and PBLs 29.5 +_ 3.5% B cells
parameters were similar
comprised
27.5 +_ 0.5% B
(Table 3).
B and T cell
for nonenhanced tumor-bearers
and
35
B. P e r i p h e r a l Blood
Leukocytes
H e l p e r S u p p r e s s o r T Cel l Rat i o
A. S p l e n o c y t e s
12
16 20
4
T r e a t m e n t Da y
12
16
20
FIGURE 2.
HELPER : SUPPRESSOR
T CELL
RATIOS
FOR "SERUM
-ENHANCED" TUMOR - BEARING RATS AND N ONEN HA NCE D TUMOR
BEARING RATS.
Ten thousand
4bs
cells were injected
i.v . into twenty male BD-IV
rats.
Two hours later,
ten control
rats
were
injected with
0.2ml NRSa
(nonenhanced tumor-bearing
rats ,0--- O---- 0)
and ten
experimental rats with
0.2ml TBSa
(enhanced
tumorbearing rats,#----•----•) .
Sera injections
continued
every second day.
Every four days, two rats from each
group and an age/sex matched nontumor-bearing
control
rat were assessed for helper and suppressor T cells by
indirect immunofluorescence.
aNRS = serum from normal rats; TBS = serum from tumorbearing rats.
36
naive,
nine
nontumor-bearing
percent of the
nontumor-bearing
percentage
rats as shown
splenocytes
rats
were
T
in
nonenhanced
splenocyte
PBLs
from
cel l s
(Table
3).
The
respectively
(Table
(Table 3).
rats
3).
B
was
cell
rats
was 25%
The proportion
pre parat ions
tumor-bearing
Forty-
of the
of B cells from nontumor-bearing
Cells
3.
and 53%
T
for splenocytes and 29% for PBLs
of
in Table
and
43
+
PBLs
from
and
45%,
2%
populations
from
splenocytes of nonenhanced tumor bearers was 29.5% and PBLs
was 30%.
(Table 3.)
Injections
as
outlined
for
of TBS,
using
Tables
2 and
the
same treatment
3,
did
schedule
not
significantly
modulate T cells in a nontumor-bearing rat.
The H :S ratio
for splenocytes and PBLs from this rat
TBS
injections
respectively
from
the
every
second
day,
(data not shown).
spleen
and
fourteen days after
were
1.52
The percentage
peripheral
blood
was
and
1.38,
of T cells
56%
and
50%,
respectively (data not shown).
BINDING PARAMETERS OF SERUM IMMUNOGLOBULIN TO LYMPHOCYTES
Splenocytes
from
rats
shown
numbers
in
from
without
Table
of cells
4,
rats bearing
tumors,
there
able
from nontumor-bearing
bound
was
subcutaneous tumors and
IgG present
little
to bind IgG;
rats
compared to 62% binding
in TBS.
difference
in
As
the
65% of the splenocytes
incubated
with
TBS
by tumor-bearing rats.
bound
IgG
The IgG
37
TABLE 3.
T AND B LYMPHOCYTE
COUNTS FOR "SERUM-ENHANCED"
TUMOR-BEARING
RATS,
NONENHANCED
TUMOR-BEARING RATS
AND NONTUMOR-BEARING RATS
Splenocytes
% B Cells
% T Cells
(+ S. E'. ) '
NontumorBearing Rats
Peripheral Blood
Leukocytes
% B Cells
K T Cells
(+ S .E . )
25
49
29
53
Nonenhanced
Tumor-Bearing
Rats (n = 2)
29.5 +_ 2.5
43 +_ 2
30.0 + 0
45 + 0
Enhanced Tumor
-Bearing Rats
(n = 2)
27.5 + 0.5
50 + I
29.5 + 3.5
49 + I
(n
= I)
Ten thousand 4bs cells were injected i .v . into four
BD-IU rats.
Two hours later, two rats were injected i.p.
with 0.2ml N R S a (n onenhanced tumor-bearing rats) and the
remaining two rats were injected with 0.2ml TBSa (enhanced
tumor-bearing rats).
Sera injections were continued every
second day until day fourteen, when the animals were killed
and assessed for changes in lymphocyte populations by
indirect immunofluorescence.
a NRS = serum
bear i n g rats.
from normal rats;
TBS = serum
from tumor-
38
binding
patterns
for
nylon
wool-passed
nonadherent
splenocytes and thymocytes indicated that the cells binding
immunoglobulin
corroborate
were
data
I lymphocytes
obtained
from
(Table
4).
These
F ACS-sorted
data
sp lenocytes.
One hundred percent of the splenocytes sorted on the basis
of IgG
binding
were
shown to be T lymphocytes by indirect
immunofluorescence.
Data
from
splenocyte
cells.
Table
or
3 indicates
peripheral
Fe receptors
that
leukocyte
(FcRs)
30%
of
the total
populations
on B cells may
were
account
B
for a
large proportion of the nonspecific binding as indicated by
the
amount
antibody
At
of
binding
by
the
F I TC-conjugated
incubated with CM only,
sub optimal
conjugated
dilutions
goat anti-rat
of
not with serum
either
the
IgG antibody,
TBS
second
(Table 4).
or
the FITC-
the immunoglobulin
binding pattern was lost.
When
and
it was
discovered
TBS
bound
to
bearing
rats,
it was
binding
patterns
bearing rats.
5.
a population
were
next
immunoglobulin
of lymphocytes
from
investigated whether
altered
for
from NRS
tumor-
lymphocyte
"serum-enhanced"
tumo r­
The results of this study are shown in Table
Binding patterns observed for PBLs removed from "serum-
enhanced"
rats
that
and
tumo r-b ear ing
naive,
control
rats,
nonenhanced
rats,
indicate
bound IgG from TBS than from NRS.
tumor-bearing
that
more
cells
PBL binding patterns for
39
TABLE 4.
BINDING
DF
SERUM
IMMUNOGLOBULIN
FROM
TUMOR­
BEARING
RATS
TO
LYMPHOCYTE POPULATIONS FROM TUMOR­
BEARING RATS AND NONTUMOR-BEARING RATS
Cells Exhibiting Positive Fluorescence
Thymocytes
Unfractionated
Splenocytes
Primary
reaction with:
CM NRS TBS
CM NRS TBS
NontumorBearing Rats
16
22
82
35
21
65
3
28
56
TumorBearing Rats
16
18
88
50
52
62
32
39
65
Nylon wool
Nonadherent
Spl enocytes
CM NRS TBSa
The 4 b s t u m o r was t r a n s p l a n t e d s u b c u t a n e o u s l y .
Nineteen days after tumor transplant, t h y m o c y t e s and
splenocytes from tumor-bearing rats and two age/sex matched
control rats were analyzed.
aCM = complete medium; NRS = serum
TBS = serum from tumor-bearing rats.
from normal rats;
40
CM,
NRS
and TBS
from
"serum-enhanced"
were 44%, 52% and 83% respectively,
tumor-bearing
rats
from nonenhanced tumor-
bearing rats 48%, 45% and 61% respectively, and from naive,
control
rats
67%,
69%
and
92%
respectively
(Table
5).
Comparable results were obtained for nylon wool nonadherent
splenocytes removed from the same rats (Table 5).
was
bound
prepared
from
from
TBS by nylon wool non adherent
"serum-enhanced"
More IgG
splenocytes
tumor-bearing
rats
nylon-wool nonadherent splenocytes from nonenhanced
bearing
rats.
splenocytes
CM,
NRS
enhanced
similar
The
binding
and
TBS
percentages
33%,
tu mor- bea rin g
cell
preparations
after
46% and
65%,
(Table
5).
rats
from
incubation
5).
splenocytes
IgG
from
binding
by
with
respectively for
IgG-binding
by
nonenhanced tumor-bearing
rats was 24%, 35% and 46% for CM, NRS and TBS,
(T ab l e
tumor­
of nylon wool n onadherent
IgG from serum
were
than
nylon
wo ol
nontumor-bearing rats was
respectively
nonadherent
7%,
9% and 20%
for CM, NRS and TBS, respectively (Table 5).
The binding
isotypes were
patterns
also
for
individual
investigated.
immunoglobulin
These data are presented
in Table 6.
Since no increase in the percent cells bearing
I g G 2 b could
be detected
PBLs
incubated
with CM,
for PBLs
incubated
"serum-enhanced"
with T BS over
tumor
bearers
appear to already have IgG2b bound to all potential binding
TABLE 5.
INTERACTIONS OF SEROM IgG
FROM
TOMOR-BEARING RATS
WITH
LYMPHOCYTE
POPULATIONS FROM
"SE ROM-ENHANCED" TU MO R-BE AR ING RATS, NONENHANCED
TUMORBEARING RATS AND NONTUMOR-BEARING RATS.
% Cells Exhibiting Positive Fluorescence
Primary
reaction with:
Nontumorbear ing rats
Nonenhanced
tumor-bearing
rats
Enhanced tumorbearing rats
Thymocytes
Un fractionated
Splenocytes
Nylon-wool
Nonadher en t
Splenocytes
Peripheral
Blood
Leukocytes
CM NRS TBS
CM NRS TBS
CM NRS TBS
CM NRS TBS
I
18
91
26
34
75
7
9
20
67
69
92
2
11
43
55
47
61
24
35
46
48
45
61
2
10
84
53
59
52
33
46
65
44
52
83
Ten thousand 4bs tumor cells were
injected i.v. into
four male BD-IV
rats.
Two hours later, two rats were injected with NRSa (nonenhanced tumor-bearing rats)
and two
rats were injected
with 0.2 ml TBSa (enhanced tumor-bearing rats).
Sera
injections
were
continued every
second
day.
On day
fourteen,
thymocyte,
splenocyte and PBL cell
populations
from rats from both treatment groups
and
age/sex matched controls were isolated and examined by the FACS.
a CM = com ple te medium,
tumor bearing rats.
NRS = serum from normal
rats and TBS = serum from
42
sites
on
P BL s .
The
percentage
of
PBLs
binding
following incubation with CM,
NRS and TBS was 74%,
71% respectively
These
binding
sites
killing
the
analysis.
for
animals
from
tumor-bearing
able
still
to
were
was
(Table 6).
to
IgGgb
not
PBLs
bind
following
that the
prior
the
for
FACS
for
PBLs
cells
rep eat ed
and
to
nonenhanced
from n on tumor-bear i n g
rats
IgGgb•
71% and
saturated
nontumor -bear ing
tumor-bea rin g
able
data indicate
preparing
phenomenon
rats
bind
isotype
and
naive,
nonenhanced
cells
this
This
prepared
and
(Table 6).
IgGgb
contained
additional
The percentage
incubation with
CM,
of PBLs
NRS and
TBS for nonenhanced tumor-bearing rats was 46%, 45% and 59%
respectively and for nontumor-bearing controls was 62%, 64%
and 85%, respectively (Table 6).
Binding of other immunoglobulin isotypes by peripheral
blood
leukocytes
was minimal.
very
or
low
nonenhanced
immunoglobulin
from
not
rats
isotypes
and
nonenhanced tumor
but binding by IgM, IgGj
demonstrated.
tumor-bearing
naive
I g G g a , IgGgb
tumor-bearing
As shown in Table 6, PBLs were able to bind
levels of IgGga iji vivo
I g G g c was
PBLs
from "serum-enhanced"
rats
PBLs
rat s
from naive rats and
did
bi n d
additional
albeit at modest levels
demonstrated
Ig G g c i_n vivo
bearing rats
binding
(Table
bound
of
6).
fewer
(Table 6).
IgM,
IgGi,
PBLs
isotypes
from
iji
TABLE
6.
IM M U N O G L O B U L I N ISOTYPE BINDING PARA METER S FOR PERIPHERAL BLOOD
LEUKOCYTES FROM "SERUM -ENHANCED" TUMOR-BE AR ING
RATS, NO NE NHANCED
TUMOR­
BEARING RATS AND NONTUMOR - BEARING RATS
% Cells Exhibiting Positive Fluorescence
IgM
Primary
reaction with:
Nontunorbearing rats
Nonenhanced
tunor-bearing
rats
Enhanced tumorbearing rats
iS&a
IS§2b
Mz2c
IsG
CM NRS TBS
CM NRS TBS
CM NRS TBS
CM N^IBS
CM NRS TBSa
15 11 12
15
15
9 13
62 64 85
18 14 14
44 32 55
20 13 16
17 13 14
12 10 12
46 45 59
__ _b
38 39 55
_
_ _ _b
12
74 71 74
____ __ b
18 56 78
IgG]
CM NRS TBS
_b
9 13
8 11
Ten thousand 4bs cells
were injected I. v. into four BD-IV male rats.
Two
hours later, two rats were injected with NRSa (nonenhanced tumor-bearing rats) and
two rats were injected with TBSa (enhanced tumor-bearing rats).
Sera injections
continued
every
second
day until
day fourteen.
On day fourteen, the PBL cell
populations from rats from both treatment groups and age/sex matched controls were
isolated and examined by the FACS.
aCM = complete medium,
tumor-bearing rats.
NRS = serum from
normal rats and
bFluoresc ence intensity was not measurable.
TBS = serum
from
44
in vivoj
binding was demonstrated for IgM,
IgGj, IgGga and
IgGgb and not for IgGgc .
MODUL AT IO N
OF
TUMOR
LUNG
COLONIZATION
WITH
ADOPTIVELY
TRANSFERRED FACS-SORTED TUMOR BEARER SPLENOCYTES
Previously, it was shown
was
enhanced
colony
I).
by
chronic
assay and
splenocytes.
injection
the spontaneous
Metastasis
transferring
that
was
also
FACS-sorted,
tumor
lung
of TBS using
metastasis
enhanced
IgG
metastasis
the
assay
by
lung
(Table
adoptively-
binding,
tumor
bearer
The average number of tumor lung nodules for
rats receiving FACS-sorted splenocytes was 77.4 compared to
43.3 for rats receiving normal saline (Table 7).
Histologic
examination
revealed
that
adoptive
transfer
tissue
of
the rats whose
had miliary
sur rounding
advanced
repr esent ative
tumor
nodules.
was
not
observed in stained
by
in the lung
The miliary
metastases were too small and too numerous to count.
phenomenon
sets
growth was enhanced
micrometastases
tumor
lung
This
sections of lungs
from the control rats (Figure 3).
At the time of injection of FACS-sorted tumor-bearing
splenocytes,
for
sorted
expression
and unsorted
of
cell
immunofluorescence.
One
splenocytes were analyzed
surface
hundred
antigens
percent
of
by
indirect
the
sorted
cells expressed the OX-19 antigen on the cell surface (data
45
TABLE 7. MODULATION
OF
ADOPTIVE
TRANSFER
SPLENOCYTES
Rat Group
Controls
(n = 5)
TUMOR
LUNG COLONIZATION
OF FACS-SORTED
TUMOR
BY THE
BEARER
Mean Number of Pulmonary
Tumor Colonies, (range), Pa
(saline)
43.3 (23-60)
Experimentals (FACS-sorted
tumor-bearer splenocytes)
(n = 5 )
77.4 (67-83)
P = 0.0079
BD-IV rats were injected with I x IO^ 4bs cells
Two hours after the tumor cell injection, control
r e c e i v e d 0 . 2 m l n o r m a l s a l i n e , and experimental
received 5 x 10^ FACS-sorted^ tumor bearer splenocytes
Animals were necropsied twenty -four days later.,
i.v.
rats
rats
i.p.
aP values were calculated using the Mann-Whitney U
Two-Tailed Test.
b I g G - po s i t i v e tumor bearer splenocytes were, sorted
using the same gates and parameters that were used to
c a l c u l a t e p e r c e n t a g e of c e l l s e x h i b i t i n g p o s i t i v e
fluorescence in previous experiments (Tables 4-6).
46
FIGURE 3.
SECTION OF LUNG TISSUE FROM A RAT
INJECTED WITH
4b s CELLS I .V. AND FACS-SORTED
SPLENOCYTES I.P.
The
section was stained with
hematoxylin
and e os in (40 Ox
magnification) and
the
arrows
point
to a miliary
micrometastatic tumor colony.
47
not
shown).
Therefore,
IgG
binding
were
the cells
sorted on the
I lymphocytes .
sorted cells was 0.57
The
(data not shown).
basis
H :S ratio
of
for the
Referring back' to
Table 2, the H :S ratio for splenocytes taken from enhanced
tumor-bearing
it was
rats
on day 14 was
0.84 +_ 0.16 and
By comparison, 6 3 . 6 %
0.57 +_ 0.03.
for PBLs
of the unsorted
tumor bearer splenocytes were T cells and the H :S ratio was
0.95 (data not shown).
Fluor escence
sorted
and
splenocyte
the
IgG
binding
which
from
displayed
were
the
TBS
IgG positively-
revealed
tumor
different
bearer
immunoglobulin
Cells sorted as positive on the basis of
fluorescently-labeled
Cells
of
negatively-sorted
preparations
binding patterns.
Ig G
microscopy
TBS
appeared
surface
sorted
a pe rip h e r a l
f l u o r e s c e n t l y - labeled
to
components
as negative
(data
pattern
patching
and
the
not shown).
for binding
fluorescent
complexes
internalize
TBS IgG
wi th
the
capping off
(data not shown).
Confi rmation
bound
of the
immunoglobulin
apparent
complexes
in
internalization of the
the "positively-sorted"
cells was sought by investigating the ability of trypsin to
remove
time
the membrane-bound complexes.
frame
the
surface
complexes
externally added trypsin, confirming
(data not shown).
After
were
a very
short
unavailable
to
their internalization
Since the complexes
were internalized
48
there was no ready method of removing them from FACS-sorted
splenocyte's.
wi t h
the
However,
ability
splenocytes
from
colonization
Fab
of
the
did not
appear
FACS-sorted,
tumor-bearing rats
to interfere
IgG-binding
to enhance tumor
prepared from
PBLs from tumor-bearing
whether
T BS were
incubated
rats and nontumor-bearing
or
not
the
Fe
analysis,
not
shown).
of
to
t he
As shown
the binding patterns were not altered by
incubating TBS fragments with
(data
with
rats,
portion
immunoglobulin was required for TBS IgG binding.
by FACS
lung
(Table 7).
fragments
ascertain
FITC
PBLs
On the other
from tumor-bearing rats
hand,
the binding pattern
for TBS fragments was lost after incubation with PBLs
nontumor-bearing rats
(data not shown).
Therefore,
from
it can
be concluded that the Fe portion of the immunoglobulin was
not
involved
in
lymphocytes. The
the
binding
Fe portion,
of
TBS
IgG to tumor
however, may
be
bearer
involved
in
immunoglobulin binding by lymphocytes from naive, nontumorbearing rats.
49
DISCUSSION
Immunological
prolonged
survival
immun ization
directed
the
enhancement
of
a
of
a tumor
loses
against
has
achieved through
of the
the
in some
antigens,
tumor and
immune system
the primary
host
tumor
instances
destroy
and
(15-20, 22-33)
(21,24).
to
by
with
the
passive
antibodies
an effective
it.
manipulation
(15-33)
refers
As a consequence,
its ability to mount
response
been
tumors
facilitated
tum or-bearing
against the tumor's
host
of
Enhancement
of the
includes
as well
immune
humoral arm
enhancement
of
as its
metastases
In the experiments
reported
herein the process of tumor metastasis has been enhanced by
chronic
injection
spontaneous
colony
tumor
tumor
metastasis
assay
in
of
the
(Table
bearer
assay
I).
The
spontaneous
and
serum
the
growth
in
both
the
experimental
lung
rate of the primary
metastasis
assay
was
not
significantly affected (Figure I).
These
ex per i m e n t s
Starkey et a I . (21)
the
exper imental
Starkey et al.
4bs
tumor
support
the
earlier
results
as well as KaIiss et al . (22).
lung
colony
assay,
previous
of
Using
work by
(21) had shown that lung colonization of the
ce ll
injections of the
line
was
enhanced
by
IgG2b fraction of tumor
thrice
weekly
bearer serum.
50
Kaliss
et al.
C57BL/K
(22) passively
mice
alloantiserum,
the
work
enhanced tumor
using
the
intact
IgG and
of Sacchi
current findings.
F (a b 1 )2 fraction
et al.
whole
(24)
of
partially
in
anti-tumor
alloantiserum.
Also,
agrees with the
Using a monoclonal antibody prepared to
Lewis Lung Carcinoma antigens,
Sacchi et al.
lung metastasis
the
growth
TBS
did
but restricted
t u m o r . In
the
current
study,
affect
growth rate
of the
the
allografts
of
not
the
primary
significantly
primary , subcutaneous
tumor but did enhance metastasis
The demonstration
(24) enhanced
4bs
(Figure I).
that metastasis
could
be
enhanced
using the spontaneous metastasis assay satisfied objections
which had been
raised against using the lung colony assay
as the only assay for assessing immunologic changes related
to tumor growth.
Injecting
tumor
cells directly into the
veins of animals does not accurately mirror events in human
cancer.
tumor
Cancer
which,
patients
over
develop
a period
of
a spontaneous
time,
may. progress
metastasize to distant sites of the body.
metastasis
primary
assay
reflects
tumor is perceived by
However, when
animal
better
times
difficulties
The
the manner
its host's
in
immune
when
after
develops metas tases
tumor
inoculation,
quantitating
changes
and
spontaneous
which a
system.
using the spontaneous metastasis assay,
in the experiment
different
primary
at
each
slightly
and this poses
influenced
by
51
treatment
modalities.
Use
removes this variability since
of the lung
colony
assay
tumor cells enter the blood
stream of each rat at the start of the experiment via i.v.
injection.
Therefore,
the lung colony assay was chosen for
the remainder of the current studies.
Enhanced
metast ati c
tumor
growth
changes in immunologic parameters.
tumor
cell
enhanced
injection,
tumor-bearing
tumor-bearing
tumor
the
rats
injection,
tumor-bearing
the
rats
H :S
paralleled
compared
2).
For
ratio
declined
by
Fourteen days after 4bs
PBL H :S ratio was
rats
(Table
was
to
depressed
in
nonenhanced
twenty days following
for
"serum-enhanced"
rapidly
(Figure
2).
In
contrast, the H :S ratios for nonenhanced tumor-bearing rats
transiently peaked on day 4 and declined thereafter (Figure
2 ).
The
H :S ratio
almost inverted
in
the
single
fourteen
post
difference
for
enhanced
tumor
bearing
rats was
compared to nonenhanced tumor-bearing
helper :suppressor
assay
tumor
(Table
in the
inoculation
H :S ratios
between
performed
2).
rats
on day
This
big
"serum-enhanced" and
nonenhanced tumor-bearing groups was not seen on either day
thirteen
experiment
or
sixteen
in
since in the
the
latter
multiple
tim e
experiment
the
point
H :S
H :S ratio
dropped for the nonenhanced tumor bearing group (Figure 2).
At least two
factors may
account
for
this
discrepancy.
52
First,
to
tumor
populations
continual
months
change
between
experiments
(88,89).
the
were
are heterogenous
time
subject
There
i/uas a lapse of six
the
helper!suppressor
tha t
initiated
and are
and
completed.
During
this
time, the 4bs tumor could have undergone phenotypic changes
which may have led to a slightly altered immune response to
the tumor.
in
the
Second,
proper
the rats may not have been
SPF
environment.
stained histologic sections,
from animals
the
single
time
point
suggested
that
animals
infectious
agents
during
nature
these
of
agent which
to,
or
change
in
would stimulate
some
immunological
of
the
this
the
H & E
from lung sets taken
experiment
colony
time.
not
results
tumor enhancement.
were
identified.
tumor
(Table
exposed
Unfortunately,
the immune
following
of
finished immediately after
H :S ratio
agents was
immediately
Examination
prepared
for the experiment
maintained
to
the
However,
system
2),
any
just
prior
cell injection,
could
obtained
in
studies
Even though
of
the animals
appeared to be healthy at the time of tumor cell injection,
exposure
of the
rats
to
a potential
pathogen
could
have
measurable effects on the immune system.
No
significant
proportion
peripheral
of
overall
B and
T lymphocytes
blood of enhanced
rats (Table 3).
changes
were observed
in
the
for the
spl ee ns
and
and nonenhanced tumor-bearing
Even though changes were not seen in the
53
percentage of B cells,
for one
or more
of another
observing
two
B cell
possibility of clonal expansion
clones
clone(s ) could
changes
and
and a simultaneous
not be
in B and
tumor-bea rin g
helper
the
excluded.
I cell
suppressor
I cell
Rather
populations
g r o u p s , differences
decrease
between the
occurred
populations
than
in
the
reflected by the
depressed H :S ratios found for enhanced tumor-bearing rats.
These
results
contradict
those of Koyama
et a l . (68) who
studied T and B lymphocytes in canine lymphosarcoma.
found
markedly
lymphocyte
lymph
increased
counts
nodes
in
(68).
B lymphocyte
the
peripheral
H o w e v e r , Koyama
and
They
decreased
blood,
et al.
spleen
(68)
T
and
did not
distinguish between neoplastic and normal lymphocytes which
may explain
research
and our
lymphocyte
other
the differences in results obtained
current
subsets
diseases
nephropathy
have
been
including
(90),
study.
IgG
systemic
Alterations
documented
nephropathy
pathways.
or
of
These are:
surfaces
by
transplantation
hematogeno us
tumor
B and
and
T
with
membranous
(SLE)
(65)
(64).
can occur
via several
the direct seeding of body cavities
penetration
into
natural
by mechanical mean s, or
metastasis
"serum-enhanced"
cells
in
patients
lupus. erythematosus
and acquired immune deficiency syndrome
Disse mi na tio n
for
from their
(91).
by
open
lymphatic
The immune systems
tumor-bearing rats were
fields,
and
of the
likely depressed
54
soon after tumor cell injection as indicated by a decreased
number
of
helper
suppressor
I
I cells.
cells
and
an
increased
Suppression
of
the
number
mediator
of
immune
cell functions such as down-regulation of helper I cells by
suppressor
I
cells
may
hav e
allowed
outgrowth
of
extravasated tumor emboli.
In
addition
correlate
growth,
with
to
the
declining
immunologic
changes
in
using FACS analysis.
to
lymphocytes
tumor-bearing
generally,
more
than from NRS.
TBS
IgG
were
expressed
suppression and
immunoglo bulin
populations from enhanced
bind
IgG
by
tumor
lymphocyte
rats was documented
Immunoglobulin from TBS was found to
from
rats
"serum-enhanced"
and
or nonenhanced
nontumor-bearing
lymphocytes bound
rats,
immunoglobulin
and
from
TBS
The population of lymphocytes labelled with
T cells;
the
O X -8
present in the
their
which may
enhanced
binding
tumor bearer
a majorit y
antigen
indicative of suppressor T cells.
were
H :S ratios
binding
on
of
these
t h eir
However,
T lymphocytes sorted
but
at
one-half
the
cells
cell
helper
also
surface
T cells
on the basis
value
of
usually
determined for naive, non-tumor bearing rats or nonenhanced
tumor-bearing rats.
have
al.
been
(92)
done
using
incubated
Similar immunoglobulin binding studies
sera
SLE
from
ser u m
SLE patients.
with
Edwards
lymphocytes
et.
from
healthy patients and sorted the lymphocytes on the basis of
55
IgG binding.
were
Ninety-seven percent of the sorted cells
I lymphocytes,
30% were
of which 70% were helper T
suppressor
I cells
100% of the sorted
the results
analysis
the
m aj or ity
suppressor
remainder
In the
of
(92)
from SLE patients.
the c e l l s
T
cells.
the
O X -8
of the
current
cells were T cells which
obtained by Edwards et al.
of serum
expressed
(92).
which
Sixty
anti gen
bou n d
on
of
the i r
cells expressed
study,
is similar
in their
On the
percent
cells and
IgG were
sorted cells
surface
and
the W3/25 antigen.
results are opposite to what Edwards et al.
FACS
other hand,
TBS
the
to
the
These
(92) reported.
The overall immunoglobulin binding patterns were quite
similar
for
lym phocytes
removed
from
tumor-bearing
and
nontumor-bearing rats in the initial FACS experiment (Table
4).
These
similarities
analyses
(Table
rats
the
for
5 and
initial
were
6 ).
less
Lymphocytes
FACS
experiment
removed nineteen days after tumor
for
experiments
(Tables
evident
5 and
days post tumor cell injection.
in
later
from tumor
(Table
FACS
bearer
4) were
inoculation; lymphocytes
6 ) were
removed
fourteen
During the later stages of
tumor growth, there is a splenomegaly caused by an increase
of neutrophils
Since
in the
neutrophils
spleens of tumor-bearing
possess
portion of immunoglobulins
surface
(94),
an
receptors
increased
rats
for
(93).
the
number
Fe
of
56
neutrophils could cause a shift in the number of cells able
to bind immunoglobulin.
IgG2b was
enhanced
vivo .
the
pre dominant
tumor-bearing
Significant
demonstrated.
PBLs
binding
Previously,
that this was the enhancing
the
same
rat
effector
cells
against
to
may
serve
or suppressor
could
directly
cytotoxic
it w as
by
other
Starkey
cells.
bound
in
iso types
et al.
model.
the
to
likely
was
in TBS,
I c e ll s.
isotype
for
In this case,
activating
suppressor T
PBLs
activity
block effector
Preferential
by
was
not
using
I g G2b binding and
signal
or indirectly
not
(21) had shown
antibody isotype
as
bound
down-regulate the cytotoxic
tumor cells,
particular
and
he pat oca rci nom a
internalization
imm unoglobulin
binding
signals
of
one
demonstrated
for
nonenhaneed tumor-bearing rats or nontumor-bearing, control
rats.
One of the problems in these kinds of flow cytometric
analyses was distinguishing clearly
binding by T cells and B cells.
had
previously
binding
electronically
the
computer.
that
immunoglo bul in
overlapped.
using
shown
I
by
FA CS - reso Iv abIe
two
to
out B cells
D IS P 4 program
immunoglobulin
Stout and Herzenberg
these
attempted
gating
Binding
the
between
carrying
available
with
parameters for the
peaks
lymphocyte
circumvent
(95)
for
subsets
this
by
immunoglobulin
the
Consort
incubation
40
of TBS
57
with nylon wool-passed
preformed
splenocytes
on the basis of TBS
and sorting experiments
IgG binding indicated
the 100% of the lymphocytes binding
TBS
IgG in the
that
chosen
cell population were indeed T cells.
Immunoglobulin
bearing
tumor
animals
binding
could
be
antigen-specific
evidence
indicates
by T lymphocytes
attributed
cell
that the
since Fab fragments
bound
tumor
(96)
bearer
described
a
surface
blocks
PBLs.
antigen
is also present
has
been
not
T , NK
Our
antigen
and
anti-human
(LFA-I)
K cell
antibody,
activity.
lymphocytes
This
(96) but it
Antibodies
against cell surface antigens have been shown
to inhibit T
cell function
If a similar
receptor
in experimental
exists
on
interaction
cytotoxic
rat
IgG still
Miedema et a I.
antibody,
on murine
for
or
lymphocytes.
receptor
described
from TBS
Previously,
monoclonal
cytotoxic
receptors
receptors.
prepared
lymp ho cy te -f un ct ion -a sso ciat ed
which
Fe
tumor-
binding of TBS IgG is not FcR
dependent
to
to
from
T cells
systems
suppressor
could
by
(96).
T cells,
result
suppressor
in
immunoglobulin-
dow n-regulation
T cells
via
of
lymphokine
signals.
Different
binding
patterns by FACS-sorted splenocytes
were observed.
T cells bound TBS IgG and internalized the
bound complex.
On the other hand,
TBS IgG
peripherally and
non T splenocytes bound
this subsequently patched
and
58
capped
off.
TBS
s p l e n oc yt es
presumably
Fab
IgG-binding
was
resista nt
due to rapid
fragments
by
to
enhanced
gentle
trypsin
internalization.
abrogated the
to rapidly internalize,
ability
although
tumor-bearing
The
treatment
use
of the TBS
capping
of
TBS
antibodies
and patching were
not observed.
Splenocytes from tumor-bearing rats were sorted on the
basis
of
their
experimental
Earlier
tumor
results
(unpublished
from
TBS
Splenic
shown
results)
Tanaka
growth
in
T cells
to
suppressor
the
T cells
was
0.57,
which
by
equally
(42%
was
Starkey
to
and
growth
in
lung
mice
enhance
colony
suppressed
tumor-b earin g
The sorted
staining with W 3/25).
shown
in the lung
experimental
tumor
(97).
and
revealed that unsorted
rats
from
enhance
et al.
metastasis
obtained
tumor-bearing
tum o r
IgG-binding
Talmadge
splenocytes
or
enhanced
colony
have
assay.
also
a Winn-type
cells were
assay.
been
assay
by
predominantly
stained with OX-8 compared to 24%
The
H :S ratio for the sorted cells
similar
to
the
H :S ratios
for
splenocytes from enhanced tumor-bearing rats at the end of
the
H :S T cell ratio kinetics experiment
finding
that tumor-bearer
enhance
immune
functions
(Figure 2).
Our
lymphocytes suppress rather than
may explain
some
of the
failures
encountered by clinicians when they have attempted to treat
tumors using adoptive immunotherapy (10, 98-100).
59
Tumor enhancement
laboratory
models.
is not
a phenomenon
In human
patients,
restricted
circulating
to
immune
complexes,
serum-blocking factors and anti-tumor antibodies
have
implicated
been
melanoma
in the
(20,29,43,44)
and
enhanced
growth
neuroblastoma
of malignant
(31,100).
It is
not known how these factors modulate the immune system, and
numerous
theories
an ti ge n-ant ibo dy
effector
cell
have
been
complexes
functions
proposed.
may
block
(37).
Circulating
regulator
Antibody
may
cell
also
or
mask
tumor cell antigens so they can not be recognized by immune
cells (37).
Our
control
studies
of
environment
suppressor
humoral
that
T cells are involved in the
enhancement.
for s up pr es si on-t he
T cells'
do w n-reguI a tion
would
suggest
suppress
antigen
of
the
helper
immune
TBS
creates
IgG2b isotype
receptors , the
and
cytotoxic
system
and
(102).
suppressor
They
cells which
activity was
found that
were
T cells.
This
cyclophosphamide
induces
Tanaka et al.
suppression
enhances tumor growth
unrestrained
Evidence of
by Tagart et al.
trinitrophenyl-induced suppressor T
induction of anti-trinitrophenyl
(102).
to
being
allow
reported
may bind
result
tumor growth and the expression of metastasis.
similar
the
sensitive
cytotoxic
blocked
the
T cells i_n vivo
(97) found that a tumor-bearing state
of the host's
in relation
defense mechanisms and
to T cell
recruitment
60
from the
thymus.
specific
suppressive
growth
which
The recruited
activity in the
disappeared
growth (97).
T cells exhibited
early stage of tumor
at the advanced stages
In the current study,
tumor-
of
tumor
a population thymocytes
from tumor-bearing and "serum-enhanced" tumor-bearing
bound
TBS
IgG
(Tables
4
and
5).
This
rats
population
of
thymocytes from tumor-bearing animals may be similar to the
thymus-recruited T cells described by Tanaka et al. (97).
The lung
stages
of
colony assay
tumor
establishment
point
of
when tumor
the
advanced
and
antitumor
allowed
growth
the
-
i n i t i ati on ,
primary
cells
are
tumor,
found
stages of cancer,
antibodies
us to bypass
are
and
the first
promotion,
proceed
to the
in the circulation.
tumor-associated
present
in serum,
and
At
antigens
which may
favor the survival of tumor emboli in the blood stream.
In
previous studies, we showed that TBS injected only close to
the time
of tumor
cell
injection
was
ine ffective
Only chronic TBS injections enhance metastasis.
(2 1 ).
Therefore,
it is likely that only tumor outgrowth was affected in the
lung colony assay.
Additional
studies may shed light
involved in humoral tumor enhancement.
on the
mechanisms
Previously,
it has
been shown that tumor-bearing splenocytes suppress iji vitro
cytotoxic ity
assays
(60-63).
It
would
be
relevant
to
investigate whether splenocytes from enhanced tumor-bearing
61
rats
have
the
ability
to
suppress
a mixed
lymphocyte
reaction or a cytotoxic I cell response.
Also,
known whether
is involved
humoral
looking
enhancement
for clonal
enhanced
bearing
of B cell
of
tumor
rats,
expansion and/or
may prove
with
development
of
system
the
Plaque
clonal
current
results
methods
so as to avoid
tumor
for
may
in
assays,
depression
to nonenhanced
to be interesting.
successful
favoring metastasis.
clones
growth.
tumor-bearing rats compared
together
immune
regulation
it is not
in
tumor-
These studies
le a d
to
the
manipulating
the
enhancement
responses
62
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MONTANA STATE UNIVERSITY LIBRARIES
stks N378.As51
Humoral enhancement o f metastasis:
RL
3 1762 00514932 I
N378
Ag51
con.2
DATE
Aslakson, Cheryl J.
Humoral enchancement
of metastasis
ISSUED TO
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N378
As 51
cop .2