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ICANCER RESEARCH 31, 864—867,June 1971)
Phytohemagglutinin Unresponsiveness in Mouse Spleen Cells
Induced by Methylcholanthrene Sarcomas'
William H. Adler,2 Tomoo Takiguchi, and Richard T. Smith3
Tumor Biology Unit, Department ofPathology, University of Florida, CollegeofMedicine, Gainesville,Florida 32601
SUMMARY
The composition of spleen cell populations and the response
of spleen cells in vitro to phytohemagglutinin
(PHA) were
studied in mice bearing methyicholanthrene-induced
sarcomas
and mice immunized with membrane antigen. With the use of
discontinuous albumin density gradients to separate spleen cell
suspensions, it was found that tumor-bearing mice and
immunized
mice had a shift in their spleen cell population,
with a depletion of the dense, small lymphocyte population
and a majority representation
of a less dense, large
mononuclear cell population.
The PHA reactivity of the spleen cells from the
tumor-bearing and immunized mice was less than normal. This
finding correlated with the shift in the spleen cell population
because the less dense cell types respond minimally to PHA. In
mice in which the tumors were excised, the PHA response of
the whole spleen cell population returned to normal.
Our findings with the tumor-bearing mice suggest that a
poor response to PHA may not be indicative of an immune
deficiency but rather may demonstrate the possibility that the
mice
are
undergoing
an active
immune
response
to their
tumors. In human cancer patients, a poor peripheral
lymphocyte response to PHA and an inability to demonstrate
delayed hypersensitivity reactions have been interpreted as
indication of an immune deficiency. However, it could be
speculated that these findings in these patients may also
suggest the presence of an active immune response to their
tumors,
rather
than
an immune
deficiency.
At present,
speculation provides a different working hypothesis
evaluation of the immune status of cancer patients.
this
for the
INTRODUCTION
Several recent lines of investigation have suggested that
cell-mediated immunity is impaired in most individuals
carrying malignant tumors (13). Several parameters have been
I Supported
in
part
by
American
Cancer
Society
Grants
ACS-T-463
and hN-62-G, Florida Division of the American Cancer Society Grant
F-7-OUF, National Institute of Child Health and Human Development
Grant HD-00384, National Institute of General Medical Sciences Grant
GM-01996, and Training Grant AI-00401 in cellular immunobiohogy
from the National Institute of Allergyand Infectious Diseases.
2 Present
address:
Fort
Detrick,
Md.
21701.
3To whom requests for reprints should be addressed, at Department
of Pathology, College of Medicine, University of Florida, Gainesville,
Fla. 32601.
Received January
864
8, 197l;accepted
February
23, 1971.
measured, including lowered absolute lymphocyte levels (12),
lessened lymphocyte responses to mitogenic stimulation (5,
16), and decreased capacity to be sensitized by chemical
agents
such
as
2,4-dinitrochlorobenzene
(3)
or
2 ,4-dinitrofluorobenzene
(8).
Moreover,
the
association
of
immunological deficiency with neoplastic disorders has been
most clear in those individuals who have abnormal or deficient
function of their thymus-dependent lymphoid system (6). It
has been tempting to link these observations causally in
general support of a hypothesis that immune surveillance has a
major role in prevention of cancer through the thymus-de
pendent,
cell-mediated
immune
mechanism
(15).
For exploration of the mechanisms underlying decreased
manifestations of cell-mediated immunity in tumor-carrying
animals, in vitro lymphocyte stimulation by PHA4 was assayed
in the spleen cells from methylcholanthrene sarcoma-bearing
mice, and the results were compared with the PHA response in
the spleen cells of animals immunized with a nonneoplastic
membrane antigenic stimulus, either sheep RBC or allogeneic
cells. The results suggest that decreased PHA reactivity found
in the spleen cell population of tumor-bearing animals may be
related
to
immunological
commitment
rather
than
immunological deficiency.
MATERIALS AND METHODS
A/J and CBA strains of mice used in these experiments were
originally
purchased
from R. B. Jackson
Laboratories
and have
been maintained for 3 years in our laboratory through
subsequent inbreeding. Tumor production was accomplished
by injections of 0.1 mg methylcholanthrene in 0.1 ml olive oil
i.m. into female mice. The tumors that arose 6 to 12 weeks
later were serially passed in female A/J mice. The 3 A/J
methylcholanthrene
sarcomas used in these experiments were
designated MA-2, MA-6, and MA-7. Tumors were not used
after a 5th mouse passage. The tumor was passed by giving
recipient syngeneic mice injections of 2 X 106 dispersed tumor
cells s.c. A palpable tumor was easily demonstrated 2 weeks
after inoculation, and the mice were used for the experiments
4 weeks after the tumors appeared. Tumor removal was
accomplished by surgical excision when the tumor measured 1
cm in diameter.
The culture method used has been reported in detail
previously (2). The same serum donor *as used for all
experiments
reported here. PHA-stimulated (10 p1/tube)
cultures consisted of 15 X 106 mononuclear spleen cells in 3
4The abbreviation used is: PHA, phytohemagglutinin.
CANCER
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Methykholanthrene
density gradients. The layers of the fractionated cells were
designated: Layer A, between the 10 and 23% albumin
solutions; Layer B, between the 23 and 26% albumin; Layer C,
between 26 and 29% albumin; and Layer D, the most dense,
small lymphocyte population, between 29 and 35% albumin
solutions. The pellet was not used for culture or considered in
the enumeration of the separated populations. Each layer is
represented as the proportion of cells in that layer in relation
to the total cells recovered in all 4 layers. As previously
described,
cells found in each layer were relatively
homogeneous with regard to density in that between 73 to
95% would migrate to the same layer when subjected to a 2nd
identical gradient fractionation (1).
ml culture medium (RPM! 1640 with 5% heat-inactivated
human serum). PHA-stimulated and control cultures were
maintained
for 48 hr. Tritiated
thymidine
(1 pCi,
Schwarz/Mann, Orangeburg, N. Y., 1.9 Ci/mmole, I pCi/2 p1)
was added to each culture during the final 20 hr. At the end of
this time, the cultures were processed as described previously
(2).
The amount
of activity
in the 5% trichloroacetic
acid
precipitates was determined by liquid scintillation counting.
The results are expressed as means of 5 replicate cultures.
Individual variation of cultures from the mean was within
10%. Immunization
was accomplished
by injection
of 0.1 ml
of a 50% washed sheep RBC suspension i.v. The 2nd and 3rd
immunizing injections were given at 10-day intervals. A/i mice
immunized with alloantigen received 0.1 ml of a CBA spleen
cell suspension containing 1 X 106 leukocytes. The method of
Raidt et a!. ( 11), modified as previously described ( 1), was
used for separation of spleen cells on discontinuous albumin
RESULTS
Gradient
of the
@,.oma.2
Id
71
Distribution
of
Spleen
Cells
in
a
mononuclear
cell population
of the normal
mouse
spleen (1). The density distribution of spleen cells was
compared between mice carrying methylcholanthrene-induced
sarcomas, and normal mice. As shown in Chart 1, a shift
occurred in the proportion of cells of various density. In the
tumor-bearing animals, there was a greater proportion of the
less dense, larger cell subpopulations.
The small, dense
lymphocytes were markedly depleted.
The PHA Stimulation of Spleen Cells from Tumor-bearing
Mice. The A and B layer cells from normal mouse spleens are
affected minimally by PHA and have high background
thymidine-3 H incorporation levels (1). Since cells of these
density characteristics are dominant in tumor-bearing mice, it
was of interest to determine their relative responses to PHA
stimulation. The results shown in Table 1 are representative of
the findings in groups of mice carrying the 3 different
methylcholanthrene-induced
sarcomas. Spleen cells from these
animals have a markedly decreased responsiveness to PHA
stimulation. In the mice carrying the MA-2 sarcoma, a normal
degree of responsiveness to PHA returned after the tumor had
14,41
A
Density
Tumor-bearing Mice. A relatively dense, small lymphocyte
population, designated Layers C and D, constitutes a majority
normal
@—oma..7
6(
Tumor Effect on Spleen Cells
D
LAYERS
Chart 1. Spleen cells from A/J mice, either normal or ones carrying
methylchohanthrene-induced sarcomas of syngeneic different origin,
designated MA-2, MA-6, and MA-i, were separated by discontinuous
albumin density gradient fractionation. The resultant profile is
expressed in terms of percentage of cells found in each layer of the
gradient as compared to the total number of cells recovered in all the
layers. Layer A is the least dense population; Layer D is the most dense.
Table 1
Comparison of the PHA response ofA/J spleen cells from normal and methylcholanthrene-induced tumor-bearing mice and
of micefrom which the tumor hasbeen excised.
In these experiments, 15 X 106 spleen cells were cultured in 3 ml of medium, with or without 10 @tl
PHA, for 48 hr.
Tritiated
thymidine
was added for the final 20 hr of culture. Results are expressed as the mean cpm of 5 replicate samples
with less than 10% individual variation. Each of the groups in these series of experiments involved the use of pooled spleen
cells obtained from the spleensof at least 4 mice.Thymidine-3
H incorporation (mean cpm/culture)
incorporationaNormal
Experimental groups
A/J
A/J
A/J
A/J
a Mean
A/J
mice with
mice with
mice with
mice with
cpm
in
MA-6 tumorsb
MA-i tumors@@
MA-2 tumorsb
MA-2 tumors excisedc
the
b Mice in this group
PHA-stimulated
had a greater
cultures
than
Unstimulated
PHA-stimulated
4,872
44,332
17,704
20,693
5,626
151,023
42,931
63,951
41,326
143,846
divided
1-cm diameter
by
the
mean
transplanted,
cpm
in
the
syngeneic,
Increment of
incorporation
146,951
(—)1401
46,211
20,633
138,220
control
Ratio of
31.8
1
3.6
1.9
25.4
cultures.
methylcholanthrene-induced
sarcoma.
c Thesemice had carried a greater than 1-cmdiameter MA-2 sarcoma;the tumor wasexcised 1 month before testing.
JUNE 1971
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865
w.H.Adler,i: Takiguchi,
andR.T.Smith
been excised. Thymidine incorporation
in unstimulated
cultures of spleen cells from mice carrying the tumors was
higher than in the control
groups of mice or the groups from
which the tumor had been excised. This correlated well with
the observation that the spleen cell population shifted toward
dominant
representation
Methylcholanthrene
Tumors.
The
results
of
experiments with tumor-bearing mice were strikingly similar to
those found previously in animals immunized with sheep RBC
or allogeneic spleen cells (1). In both instances, a shift in
spleen cell subpopulation
representation
occurred,
as
demonstrated in Chart 2. In mice thus immunized, the
mitogenic stimulation of either sheep RBC stroma or of
allogeneic cells in mixed leukocyte cell culture was most
effective in the B layer cells ( 1). Raidt et al. ( 11) also found
that
the B layer contained
a high proportion
of
antibody-forming cells.
a
sheep RBC-immunized and ailogeneic cell-immunized mice was
examined.
of A and B layer types of cells.
Comparison of Effects of Immunization with the Effects of
Bearing
PHA Reactivity of Spleen Cells from Either Sheep RBC- or
Allogeneic
Cell-immunized
Mice. Since the spleens of the
immunized
mice contain more B layer cells and less small,
dense lymphocytes,
the PHA reactivity of the spleen cells from
___normal
G@O
imraunizad
Mice that had received
either
I or 3 immunizing
injections of sheep RBC i.v. showed changes both in degree of
background
unstimulated
thymidine
incorporation
and
stimulation
by PHA (Table 2). This change was less
pronounced in the group immunized with allogeneic cells. As
in tumor-bearing mice, a shift in spleen cell population toward
greater representation of B layer cells was associated with
higher background incorporation of tritiated thymidine and
impaired reactivity to PHA.
DISCUSSION
In both human and animal hosts, cell-mediated immunity
and antibody with specificity toward tumor-unique antigens
have been demonstrated in many systems. Moreover, the
immune
state of the host appears to have a role in determining
the outcome of the host-tumor interactions. Experimental
tumor rejection appears to be mediated most effectively by
immune cells, although whether through cell-mediated or
antibody secretion mechanisms is not resolved (1 5). Hellström
et al. (7) found evidence that a major component
of the
A
I
C
D
LAYERS
Chart 2. Spleen cells from CBA mice, normal and immunized with
sheep RBC, were separated by discontinuous albumin density gradient
fractionation. The profile is expressed as the percentage of cells found
in each layer of the gradient as compared to the total number of cells
recovered
in all layers. Layer A is the least dense population;
Layer D is
the most dense.
immune response to a tumor, a blocking factor, presumably
antibody, can be associated with faster progress of the tumor
to the detriment of the host. This antibody-like factor can
inhibit the in vitro tumor recognition response of host
lymphocytes.
Our present studies demonstrate that the PHA reactivity of
mouse spleen cells is markedly affected by the state of the
immune mechanisms of the animals. Whether the animal was
marshalling
an immune
response
to sheep
RBC or allogeneic
cells or putatively to a methylcholanthrene
tumor, similar
changes occur. The PHA-reactive cell subpopulation of the
spleen, mainly small lymphocytes,
decrease in their
proportional
representation
in the spleen, and the total
Table 2
Comparison of the PHA response ofA/J and CBA spleen cells from normal and sheep RBC- or alloantigen-immunized mice
In these experiments, 15 X 106 spleen cells were cultured in 3 ml of medium, with or without 10 i.@h
PHA, for 48 hr.
Tritiated thymidine was added for the final 20 hr of culture. Results are expressed as the mean cpm of 5 replicate samples
with less than 10% individual variation. Each of the groups in these series of experiments involved the use of pooled spleen
cells obtained from the spleens of at least 4 mice.Thymidine-3
H incorporation (mean cpm/culture)
incorporationaNormal
Experimental groups
A/J
A/J receiving 1 injection of sheep RBCb
A/J receiving 1 injection of CBA spleen cellsb
NormalCBA
CBA receiving 1 injection of sheep RBCb
CBA receiving 3 injections ofsheep RBCb
Unstimulated
i,468
50,004
32,415
7,612
26,201
24,626
PHA-stimulated
139,388
173,143
291,5 14
180,013
113,889
71,274
Increment of
incorporation
Ratio of
131,920
123,139
259,099
172,401
87,688
46,648
a Mean cpm in the PHA-stimulated
cultures divided by the mean cpm in the control cultures.
b Immunized
mice received
0.1 ml of a 50% suspension
of sheep RBC i.v. or 1 X 106 CBA
spleen
18.7
3.5
9.0
24
4.3
2.9
cells i.v., once
or at
10-day intervals. The mice were sacrificed 2 days after the last injection, and the in vitro response to PHA was assayed.
866
CANCER
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Methyicholanthrene
reactivity of the spleen cells to PHA stimulation decreases. The
cells involved in a tumor-specific, sheep RBC, or allogeneic cell
immune
response
may
be
antibody-forming
cells
or
lymphocytes dividing in the presence of membrane antigens as
occurs in a purified protein derivative-driven clone of Bacillus
Calmette-Guerin-sensitized
cells
(9).
These
stimulated
cell
populations may produce, by expansion, an actual space
occupying lesion that crowds out other potentially responsive
cells. On the other hand, there could be a functional lesion,
possibly produced by the stimulated lymphocytes, which may
secrete inhibitors in vivo analogous to the inhibitors that are
known to be elaborated in vitro by lymphoblasts (14). A
relatively rapid return to a normal response in an animal in
which the tumor was removed lends support to this idea.
Recent experiments of Ming and Ming (10) show quantitative
cellular changes in the spleens of tumor-bearing mice. Their
studies were interpreted to suggest that a major shift in such
spleens was toward antibody-producing
cells or their
precursors.
These findings permit an alternative interpretation of the
PHA unresponsiveness of peripheral lymphocytes from cancer
patients and the inability to sensitize these patients with
chemical
compounds.
Instead
of
reflecting
a
state
of
immunological deficiency in any absolute terms, the failure to
sensitize tumor patients may reflect evidence of an intensive
immune response, especially an antibody response, to his
tumor. Hellström's data and previous studies of the
phenomenon of enhancement (4) suggest that this might be
detrimental to the host and for this reason might correlate
with a relatively poor prognosis. As a working hypothesis, it
could be suggested that the inability to sensitize a patient to
obtain a delayed hypersensitive response or a poor peripheral
lymphocyte response to PHA in vitro may indicate a high
degree of immune commitment to the tumor rather than an
immune deficiency state. This hypothesis would suggest a
different approach in the evaluation of the immune status of a
cancer patient.
REFERENCES
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PPD, Allogeneic Cells and Sheep Erythrocytes on Albumin
Gradient Fractionated Mouse Spleen Cell Populations. Cell
Immunol.,
1.78—91,
1970.
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Recognition by Mouse Lymphocytes in Vitro. I. Definition of a
New Technique and Results of Stimulation by Phytohemagghutinin
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ACKNOWLEDGMENTS
Tumor Effect on Spleen Cells
16.
Transplanted Methylcholanthrene-induced
Sarcomas. Scientific
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Immune Response. J. Expth. Med., 128. 681—698, 1968.
Riesco, A. Five-Year Cancer Cure: Relation to Total Amount of
Peripheral Lymphocytes and Neutrophils. Cancer, 25: 135—140,
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Smith, R. T., and Adler, W. H. Human Tumor Immunity. New
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Inhibitor of DNA Synthesis and a Nonspecific Mitogen Elaborated
by Human Lymphoblasts. Am. J. Pathol., 60. 495—504, 1970.
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JUNE 1971
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867
Phytohemagglutinin Unresponsiveness in Mouse Spleen Cells
Induced by Methylcholanthrene Sarcomas
William H. Adler, Tomoo Takiguchi and Richard T. Smith
Cancer Res 1971;31:864-867.
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