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[CANCER RESEARCH 39, 4564-4574, November
1979]0008-5472/79/0039-OOOOS02.00
Matching of Chemotherapy to Mouse Strain and Lymphoid Tumor Type
toPrevent Tumor-induced Suppression of Specific T- and B-Cell
Functions1
Ronald B. Faanes,2 Vincent J. Merluzzi, Neil Williams, George S.
Tarnowski, and Peter Ralph
With the technical assistance of Stephen Banks, Harry
Satterwhite, Susan Urbano, Margaret Walker, and Heather Jackson
Walker Laboratory, Memorial S/oan-Kettering Cancer Center. Rye,
New York 10580
ABSTRACT
Specific immunological and hematopoietic functions werestudied
during treatment with antineoplastic agents in micebearing
syngeneic lymphoid tumors: 702/2, a B-cell lymphoma
of C57BL x DBA/2 F, (hereafter called (BD2F,) mice; EL4, aT-cell
lymphoma of C57BL/6 mice; or J774, a macrophagetumor of BALB/c
mice. Both B- and T-lymphocyte function(antibody-forming cells and
cell-mediated lympholysis toward
alloantigens) were suppressed in spleen cells of mice
bearingthese tumors. Other hematopoietic functions
(granulocyte,macrophage, and megakaryocyte progenitor cells) were
variably influenced by growth of these lymphoid tumors.
J774enhanced, but 70Z/2 suppressed, megakaryocyte progenitorcells.
J774 and 70Z/2 increased levels of granulocyte-mac-rophage
progenitor cells. EL4, the T-cell lymphoma, did not
influence either cell type.Significant variation in strain
sensitivity to drug toxicity and
drug effectiveness in different tumor-host systems was observed.
Increased median survival time with reversal of tumor-
induced immune dysfunction, without toxicity to
hematopoieticprogenitor cells, was realized in two tumor-host-drug
combinations. Polyinosinic-polycytidylic acid was effective
againstJ774, while actinomycin D was active against 70Z/2.
Mito-
mycin C effectively reduced tumor load, as evidenced by lossof
splenic tumor colony-forming cells for all three tumors. This
agent prolonged survival and concomitantly restored
immunological responsiveness in hosts ¡mmunosuppressed by growthof
70Z/2 or J774. Paralleling tumor reduction with mitomycinC therapy,
the splenic hematopoietic progenitor and colony-forming B-cells
were reduced in tumor-bearing and tumor-free
mice, thus compromising its therapeutic
effectiveness.1-/5-D-Arabinofuranosylcytosine reduced tumor load
with
marginal toxicity toward hematopoietic progenitor and
colony-forming B-cells. However, immune responsiveness was only
partially restored, and median survival was not increased.
Theresults presented show the diversity of therapeutic drug
effectiveness in increasing mean survival time and influencing
otherlife-sustaining parameters (immunological and
hematopoietic
functions).
INTRODUCTION
Lymphoid functions have been found to be markedly sup
pressed in humans and laboratory animals bearing tumors (19,33,
40, 41, 43, 47, 48). That anticancer agents themselvesare
immunosuppressive (4, 24) results in further compromiseof host
defense during therapy for malignant disease. Thisextensive
reduction in lymphoid and hematopoietic responsiveness is a major
threat to the patient because of increasedsusceptibility to
infections which are severe and often fatal (2).
We previously reported (46) that mice bearing tumors originating
from the T-lymphocyte or macrophage compartment of
the immune system significantly suppressed the capacity ofthe
host for generating CTL3 in vitro. These tumors differed in
their susceptibility to several antineoplastic agents.
Administration of Mit-C, ara-C, or nitrogen mustard significantly
reduced
tumor load. Concomitant with reduction of tumor load,
thecapacity to generate CTL was restored, and median survivaltime
was increased.
Most defenses against infection, however, do not reside in
asingle lymphoid compartment. Humoral antibody responsesappear to
be more prone than do T-cell functions to toxic
effects of anticancer drugs (6, 16, 22, 34, 45). In this
study,we simultaneously evaluated B- and T-cell function
duringtherapy. Specificity of drug-induced splenic dysfunction
was
determined by quantitating hematopoietic capacities in
spleensduring therapy.
These studies were undertaken to find therapeutic regimesthat
would differentially reduce the lymphoid leukemia withoutmeasurable
toxicity to the normal immune or hematopoieticfunctions.
MATERIALS AND METHODS
Tumors. EL4 lymphoma (11) was maintained and tested asascites in
male C57BL/6J mice, P815-X2 mastocytoma (9) inmale DBA/2J mice, and
J774 histiocytic lymphoma (37) infemale BALB/c Crl mice. 70Z/2 was
induced with methylnitro-sourea in a thymectomized C57BL x DBA/2 F,
(hereaftercalled BD2F,) mouse (1). It contains cytoplasmic
immunoglob-ulin4 and is similar to the B-lymphoma 70Z/3 (35).
C118
myeloma of the C3H mouse was obtained from G. Dennert(Salk
Institute, La Jolla, Calif.) and maintained in Dulbecco'smodified
Eagle's medium containing 10% horse serum.
C57BL/6 and DBA/2 mice were obtained from The JacksonLaboratory,
Bar Harbor, Maine, BD2F( mice were from ARS/
1Supported in part by Grants CA-08748, CA-16271, CA-17404,
CA-18856-
2, and CA-24300 from the National Cancer Institute and by the
NIH BiomédicalResearch Grant 5S07 RR 05534.
2 To whom requests for reprints should be addressed, at Donald
S. WalkerLaboratory, Sloan-Kettering Institute for Cancer Research,
145 Boston PostRoad, Rye, N. Y. 10580.
Received April 25, 1979; accepted August 3, 1979.
3 The abbreviations used are: CTL, cytotoxic T-lymphocytes;
Mit-C. mitomycinC; ara-C, 1-/5-D-arabinofuranosylcytosine; FCS.
fetal calf serum; ß-ME,/S-mer-captoethanol; LDH, lactic
dehydrogenase; RFC, plaque-forming cells; SRBC.sheep RBC; CFU-B,
colony-forming units, B-cell precursors; CFU-M, colony-forming
units, megakaryocyte progenitor cells; CFU-C, colony-forming
units,granulocyte-macrophage progenitor cells; Act-D. actinomycin
D; poly(rl)-poly(rC), polyinosinic-polycytidylic acid; MST, median
survival time, days.
* P. W. Kincade and C. J. Paige, unpublished data.
4564 CANCER RESEARCH VOL. 39
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Therapy of Tumor-induced Splenic Immune Dysfunction
Sprague-Dawley, Madison, Wis., while the BALB/c mice were
obtained from the Charles River Breeding Laboratories,
Wilmington, Mass. Mice weighed 18 to 22 g at the time of
tumorimplantation and were given water and food ad libitum.
The number of tumor cells present in the spleens of
tumor-bearing mice was assessed by culturing 1 x 105 spleen
cells
in Falcon 1001 tissue culture plates in 1 ml of 0.3% agar
inMcCoy's tissue culture medium containing 20% PCS (46).Cultures
were incubated in a humidified incubator at 37°,and
the number of tumor colonies was counted on Day 5 (EL4),Day 7
(70Z/2), or Day 12 (J774). B-lymphocytes and 70Z/2required 5 x 10~5
M ß-MEfor colony growth, and spleen cells
from 70Z/2-bearing mice were plated with rabbit anti-IgMserum to
eliminate growth of normal B-colony-forming cells
(21). The other tumor cells grew without exogenous stimuli.The
percentage of plating efficiency of ascites tumor cells was26.8
±1.6, 13.5 ±1.1, and 14.3 ±0.9 (S.E.) for J774, EL4,and 70Z/2,
respectively.
LDH Virus. Plasma LDH levels were determined on heparin-
ized aliquots of plasma from mice bled at sacrifice. LDH
activitywas monitored, and activities were expressed as described
byRiley (38). The tumor-free mice of the strains used were con
sistently LDH free. Of the 3 tumors used in this study, only
EL4significantly increased plasma LDH activity during growth
invivo, suggesting the presence of LDH virus. Attempts to freethis
tumor line of LDH virus by passage in immunosuppressedrats or in
the tissue culture (30) failed to decrease LDH in theplasma of
EL4-bearing animals. 70Z/2 and J774 remained
LDH free throughout these studies. The tumors were free ofother
passenger viruses, as assayed by Microbiological Associates,
Bethesda, Md.
In Vitro RFC-forming Cell Assay. Mouse spleen cells were
prepared in a manner similar to that described by Mishell
andDutton (28) and cultured in Roswell Park Memorial
InstituteMedium 1640 supplemented with 10% specifically
screened,heat-inactivated FCS and 5 x 10~5 M ß-ME.Generally, 1 to
2x 107 spleen cells/ml were cultured in 35- x 10-mm (Falcon
No. 3001) plastic Petri dishes with an optimal number (5 x106)
of SRBC (Grand Island Biological Co., Grand Island, N.Y.) as
antigen. The cultures were incubated at 37° in Dutton
tissue culture chambers on a rocking platform in a
humidifiedatmosphere of 10% CO2, 7% O2, and 83% N2. The
cultureswere fed daily with medium (0.15 ml/dish), and direct
(IgM)anti-SRBC PFC were determined on the fourth day by a modi
fication of the plaque assay of Jerne ef al. (18).In Vitro CTL
Induction and Cytotoxicity Assay. The capacity
for CTL induction against allogeneic tumor cells was performedas
follows (17): 2.5 x 106 spleen cells isolated as describedabove
were mixed with 2.5 x 105 Mit-C-treated tumor cells
serving as stimulating antigen. The cell mixture (0.2 ml)
wasseeded in Linbro No. 76-014-05) round-bottom microtiter
plates and cultured in a humidified CO2 incubator. The level
ofCTL activity generated was monitored by adding 5 x 103 51Cr-
labeled target cells in 20-/il aliquots directly to the wells
aftera 5-day culture period. The contents of individual wells
weremixed and then centrifuged for 30 sec at 250 x g prior
toculture for 5 hr. The extent of target cell lysis was
determinedafter a second mixing to equilibrate released 61Cr,
followed by
a brief 450 x g centrifugation. Supernatant (0.1 ml)
fromindividual quadruplicate cultures was monitored for 51Cr
activ
ity. Lymphocyte counts were done routinely and revealed lym
phocyte-to-target cell ratios of 20 to 30/1. Percentage of
specific cytotoxicity was calculated using the
formula:Experimental 51Cr release - spontaneous 5'Cr release
Total 51Cr release - spontaneous 51Cr release x 100
Hematopoietic Colony Assays. In vitro cloning of CFU-B,CFU-M,
and CFU-C was accomplished by culturing the spleen
cells used for the immunological assays in semisolid agar.CFU-B
were counted on Day 6 from cultures of 2 x 104 spleencells
incubated with 5 x 10~5 M ß-ME,1% SRBC, and 10 figof
lipopolysaccharide in 1 ml of McCoy's tissue culture medium
containing 20% FCS and 0.3% agar (21, 27). CFU-M werecloned by
culturing 2 x 106 spleen cells in 1 ml of McCoy's
modified Medium 5A containing 15% FCS in 0.25% Bacto agarand
nutritional supplements. For CFU-M, 2 growth activities
have been shown to be required for quantitation (50).
Theseentities were obtained for these studies from concentrates
ofconditioned media from cultures of the murine
myelomonocyticleukemic cell line (WEHI-3) and from supernatants of
culturedmacrophages. The entity of WEHI-3-conditioned medium is
obligatory for colony growth, while the macroptiage supernatant
did not directly stimulate but potentiated CFU-M numbers.CFU-M were
grown in cultures containing 100 /il of concentrates of
WEHI-3-conditioned medium and 200 /il of the mac
rophage supernatant. Cultures were scored at x40 magnification
for CFU-M after 6 or 7 days of incubation at 37° in a
humidified incubator in an atmosphere of 7% CO2 in
air.Megakaryocyte colonies were readily identifiable and could
bedistinguished from other colony types by the extremely largesize
of the majority of the cells contained in them.
Cytochemicalidentification of megakaryocytes was obtained with
isolatedcolony cells by staining for the presence of
acetylcholinester-ase (32).
CFU-C were grown using the same conditions as for theCFU-M
except that 0.3% Bacto agar was used, and colony
formation was stimulated by concentrates of medium conditioned
by L-cells. Cultures were scored at day 7 (44).
Preparation of Colony-stimulating Activities. The
WEHI-3-conditioned medium was prepared from supernatants of
thecultured cells which were subcultured at 5 x 106 cells/ml in25
ml in 75-sq cm plastic flasks in McCoy's Medium 5A with2% FCS and 5
x 10~5 M ß-ME.The medium was collected
after 3 or 4 days, centrifuged at 3000 x g for 5 min, and
storedfrozen at —20°.Large volumes were concentrated 5-fold
by
filtration with Amicon PM10 filters. Since neutrophil
andmegakaryocyte stimuli were found to be preferentially diminished
by dialysis, this step was omitted. Polyethylene glycol(0.001 %)
was added to all preparations (44). A single preparation was used
in these experiments, and 100 /il of the 5-fold
concentrate was generally used.Colony-stimulating activity in
L-cell-conditioned medium was
prepared from L-cells grown in the same manner as WEHI-3.After
centrifugation, the L-cell-conditioned medium was di-
alyzed overnight against 2 changes of distilled water
andconcentrated 5-fold by filtration with Amicon PM10 filters.
This
preparation was titrated for biological activity, and
plateaulevels of L-cell-conditioned medium were used in all
experiments. Greater than 90% of all the colonies stimulated by
L-cell-conditioned medium were macrophages at Day 7 of culture, the
remainder being mixed macrophage-neutrophil colo
nies.
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R. B. Faanes et al.
Potentiating activity for megakaryocyte colony formation
wasobtained from macrophage supernatants (49). Macrophageswere
obtained from the peritoneal cavity of C57BL/6 micewhich had been
given injections of 1 ml of mineral oil i.p. 4days earlier (31).
The peritoneal cells were incubated at 37°
for 3 days in the absence of PCS in 25-sq mm Corning flasksat 2
x 106 cells/ml, after which the supernatants were re
moved, pooled, centrifuged, and assayed for potentiating
activity.
Chemotherapy. Act-D, ara-C, and Mit-C were obtained from
the Cancer Chemotherapy National Service Center, Bethesda,Md.
Poly(rl)-poly(rC) was prepared by Dr. Leonard D. Hamiltonof the
Brookhaven National Laboratory, Upton, N. Y. Chemicalswere
dissolved in pyrogen-free 0.85% NaCI solution.
Drugs were administered i.p. once a day for 6 days, startingthe
day following tumor implantation (Day 0). Tumor cells (1x 106) were
implanted i.p. into test groups consisting of 8
mice, of which 2 mice served as the source of spleen cells
forinvestigating immune and hematopoietic capacities.
Antitumoreffect was evaluated by the MST of 6 treated
tumor-bearing
mice from each group. The spleen donor animals were sacrificed
the day following the final injection. This was immediatelyafter
the end of therapy, but soon enough before untreatedtumor-bearing
mice begin to die or become moribund.
RESULTS
Suppression of Splenic T- and B-Cell Functions in MiceBearing
Syngeneic Lymphoid Tumors. We previously reported (46) that spleen
cells from mice bearing syngeneiclymphomas were significantly
reduced in their capacity toproduce CTL against alloantigens in
vitro. In this study, weinvestigated the in vitro production of
anti-SRBC IgM in parallel
with the generation of CTL. Table 1 shows that all
lymphoidtumors tested concomitantly suppress host capacity for in
vitroantibody as well as CTL responses. In contrast, the tumor
typeshad variable effects on hematopoiesis, as determined by
assessing progenitor cell levels of granulocytes, macrophages,and
megakaryocytes. Granulocytes and macrophages arisefrom a common
precursor cell, the CFU-C. CFU-M are a sep
arate precursor cell population giving rise to colonies
comprised predominantly of megakaryocytes. The T-lymphoma
(EL4) did not influence hematopoietic progenitor cell
levels(Table 2). The macrophage cell line (J774) elevated CFU-Cand
CFU-M levels. In contrast, the B-lymphoma (70Z/2) influenced the 2
progenitor cell classes differently, elevating CFU-C levels, while
markedly inhibiting CFU-M levels (Table 2).
Table 1CTL and RFC responses of spleen cells from mice bearing
T, B, or macrophage tumors
Spleen cells from normal or tumor-bearing mice were cultured
with their respective stimulator cell or SRBC. CTL and RFCactivity
were monitored as described in"Materials and Methods."
Therapeutic Effect of Mit-C and Restoration of ImmuneResponses
in Mice Bearing 70Z/2, J774, or EL4 Tumors.Treatment of mice
bearing 70Z/2 or J774 with Mit-C (Table 3)showed a dose-dependent
restoration of in vitro CTL (Column6) and anti-SRBC RFC (Column 7)
responses which parallel the
decrease in splenic tumor cells (Column 4) expressed as
colonyformation in soft agar. In the case of BD2F, mice bearing
70Z/2, a significant increase in MST was observed. Mit-C did
not
affect CTL or RFC responses in normal BD2F! or BALB/c miceuntil
doses in excess of 0.3 mg/kg/day were administered.Although CTL
responses appeared fully restored in Mit-C-treated, tumor-bearing
mice, the number of splenic RFC did not
exceed 50% of the RFC in controls. In contrast to the reducedin
vitro RFC response in animals receiving Mit-C therapy, B-
lymphocyte colony formation was unaffected by doses of up to0.3
mg/kg/day (Column 8). A dose-dependent decrease inCFU-C and CFU-M
levels was observed (Columns 9 and 10) at
higher doses of this agent.Mit-C reduced the tumor load in the
spleens of EL4-bearing
mice. However, C57BL/6 mice were very susceptible to thetoxic
effects of the drug, and all parameters measured wereinhibited by
doses of Mit-C as low as 0.1 mg/kg/day. Thus,
no concentration of this drug was found which would reducethe
tumor load and simultaneously restore immune function inthis
tumor-mouse strain combination.
Partial Restoration by ara-C of Immune Responsivenesswithout
Increase in MST. Our previous report (46) indicatedthat ara-C
reduced the total packed cell volume (ml) of EL4ascites to zero at
drug doses of 1 to 3 mg/kg/day. However,no significant increase in
MST was seen. This chemical reduced the tumor load in spleens of
mice bearing each of the 3tumors (70Z/2 not shown) and partially
restored immune parameters (Table 4). Tumor-free C57BL/6 mice did
not show
evidence of drug toxicity in the doses tested, and RFC and
CTLresponses were not significantly different from those of
thecontrols. CTL responses in normal BALB/c mice were notaffected
by ara-C; however, RFC responses were abrogated at
Table 2Effect of in vivo tumor growth on spleen CFU-C and CFU-M
levels
Results are the mean of 3 to 6 experiments.
Mean value (% of normalvalue)Tumor
CFU-CMacrophage:J774164 (88-265)"
T-lymphoma:EL4 90 (54-1 38)B-lymphoma:70Z/2
184(130-230)CFU-M173(133-228)
89(59-133)30(17-41)a
Numbers in parentheses, range.
TumorEL4
RL)
BALB/c (H-2 d)AKR (H-2 *)BD2F, (H-2d/0)BALB/c
(H-20)Stimulator
cellP815(H-2d)
EL4 (H-2 )EL4(H-2b)C118(H-2*)EL4 (H-2 °)%
specific5Tumor-free
mice37.4±2.8a
36.2 ±4.235.8 ±1.218.8
±1.175.7 ±3.2'Cr
releaseTumor-bear
ingmice
-
Therapy of Tumor-induced Splenic Immune Dysfunction
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Therapy of Tumor-induced Splenic Immune Dysfunction
doses greater than 3 mg/kg/day. This reduction in RFC response
occurred in the absence of demonstrable effects onCFU-B; however,
hematopoietic progenitor cells were reducedat drug doses above 10
mg/kg/day.
Successful Therapy of J774-bearing Mice by Poly(rl)-poly(rC)
Therapy. The most pronounced difference in responsiveness to
therapy of the 3 lymphoid tumors was observedwith the
immunomodulator poly(rl)-poly(rC). Reduction in J774splenic tumor
load was readily apparent and correlated with anincrease in MST.
This occurred while B- and T-lymphocyte
functions were restored (Table 5) in the absence of toxicity
tohematopoietic precursor cells. Splenic B- and T-lymphocyte
functions were also restored without toxicity of
hematopoieticprecursor cells (Table 5). In contrast, this agent did
not havean antitumor effect and did not effect increase in MST of
theimmune functions in EL4-bearing mice; in fact, it was quite
toxic to immunological responses in normal C57BL/6 mice.The
CFU-B were not affected, however. Poly(rl)-poly(rC) enhanced the
granulocyte-macrophage precursor levels 2- to 3-fold in spleens of
C57BL/6 normal and tumor-bearing mice at
high doses of the drug.Restoration of Immune Functions, but
Differential Re
sponses of Tumors to Act-D Therapy. Act-D greatly increasedthe
survival time of mice bearing 70Z/2 (Table 6). The increased MST
correlated with reduced tumor load in the spleenand restoration of
immune functions. Total nucleated cell recovery and hematopoietic
function were not disturbed by administration of this agent to
BD2F, mice. Act-D was ineffective
in increasing MST of mice bearing EL4 (not shown) and
J774.However, immunological functions were restored in both
strainsof mice in parallel with the elimination of tumor cells from
thespleen. This drug contrasts with ara-C, which reduced tumorload
but failed to restore CTL or RFC responses in J774-bearing
mice.
LDH Virus Involvement in Tumor-induced Suppression.Because LDH
virus, harbored in tumor cell lines and endogenous to certain mouse
strains, has been shown to be animmunosuppressive agent (30), we
felt it necessary to monitorplasma LDH levels during chemotherapy.
Replication of LDHvirus in mice after tumor implantation could
readily account forthe observed suppression of immune responses in
tumor-bearing mice. A fall in LDH levels paralleling therapy could
arguethat the immune dysfunction was a result of LDH virus
propagation rather than of tumor progression. Tables 3 to 6
showthat mouse stocks used in this study were free of LDH
virus.Mice bearing 70Z/2 and J774 failed to show elevated LDHplasma
levels, indicating that these tumors were free of thisvirus.
C57BL/6 mice, although free of LDH virus in the absenceof tumors,
showed very high plasma LDH levels in EL4-bearing
mice on the day of sacrifice (Tables 3 to 5). However,
therapywith ara-C which allowed partial recovery of B- and
T-cell
responses did not significantly alter plasma LDH levels
(Table3), suggesting that LDH virus was not the cause of
immuno-
suppression observed in these experiments.Suppression of Normal
Spleen Cell Responses in Vitro by
Syngeneic Tumor Cells. Tables 3 to 6 demonstrate thatspleens
from tumor-bearing mice had significant tumor infiltra
tion. It was possible that outgrowth of tumor cells during
cultureof tumor-bearing spleens accounted for the immune
dysfunc
tion we observed. To examine this possibility, known numbersof
viable ascitic tumor cells were added to normal CTL and
RFC induction cultures. Table 7 shows that the induction ofCTL
was readily suppressed in cultures of normal spleenswhen greater
than 1,500 EL4 or 500 J774 cells were added tothe generating
cultures. This is equivalent to 650 EL4 or 135J774 colony-forming
units when corrected for plating effi
ciency. RFC responses were not suppressed until greater
than15,000 viable EL4 or J774 cells were added. 70Z/2 cells,
incontrast, did not suppress in vitro development of either immune
function at cell doses up to 50,000 cells (7,150 colony-forming
units). Addition of in wfro-cultured tumor cells (data not
shown) produced comparable suppression of spleen cell cultures,
indicating that host cells contaminating the tumor asciteswere not
responsible for the observed suppression.
DISCUSSION
In the present study, abrogation of tumor-associated sup
pression of CTL and RFC responses accompanied therapy
withtolerated doses of some drugs (summarized in Table 8).
Significant reduction in splenic tumor load was observed in
alltumor-drug combinations except in EL4 treated with poly(rl)-
poly(rC). The cytoreductive effect of the drugs, however, wasnot
always reflected in increased MST or restored immuneresponsiveness.
The restoration of tumor-induced immune dys
function by the chemotherapeutic agents listed did not correlate
with changes in plasma LDH activity, indicating that LDHvirus was
not responsible for the suppressed immune functionsin tumor-bearing
animals.
Effect of Tumor on Immune Function and Hematopoiesis.70Z/2 tumor
cell colonies in spleens of untreated BD2F, werein the range of 6.5
x 103 colonies/106 spleen cells. Tumor
cell colonies in spleens of J774 or EL4 tumor-bearing micewere
in the range of 640 J774 colonies/106 spleen cells or1160 EL4
colonies/106 spleen cells, respectively. In eachcase, 5 x 10s
spleen cells were cultured in the CTL inductionassay, and 2 x 107
spleen cells were cultured in the anti-SRBC
culture (Tables 3 to 6). We therefore calculated that
RFCcultures containing spleen cells from untreated animals had3200
702/2, 320 J774, and 580 EL4 tumor colony-forming
cells, respectively, at the initiation of in vitro cultures. In
allcases except 70Z/2, based on these calculations, suppressionof
RFC and CTL development by tumor cell outgrowth inuntreated mice
was expected. Mit-C and ara-C, at doses which
did not affect control responses and reduced tumor below
thepredicted suppression level, did not completely restore B-
andT-cell responses. We concluded from these calculations that
tumor outgrowth in culture accounted for suppression in
untreated controls but did not explain suppression after
effectivetherapy (Table 3, Mit-C versus 70Z/2; Table 4, ara-C
versus
EL4).The studies reported here do not allow a definite
conclusion
to be drawn as to the nature of the cell being suppressed
intumor-bearing mice. It is well recognized that RFC and CTL
development require the collaboration of 3 lymphoid cell
compartments. RFC development to SRBC requires B-cells,
T-cells,
and macrophages, while CTL development requires collaboration
between 2 T-cell populations and macrophages (8, 20,39). The
tumor-induced immune dysfunction may reside in any
one of these lymphoid cell compartments. Haba et al. (14)
haveshown that antibody responses to T-dependent antigens were
suppressed in mice bearing Ehrlich ascites tumor, whereas
the
NOVEMBER 1979 4569
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-
R. B. Faanes et al.
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Therapy of Tumor-induced Splenic Immune Dysfunction
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R. B. Faanes et al.
Table 7In vitro suppression of cytotoxic T-cell and RFC
generation by tumor growthAllogeneic reactions of C57BL/6 versus
P815 (H-2°),BALB/c versus EL4
(H-2°)and BD2Fi versus C118 (H-2*) were cultured for 5 days in
vitro. The
syngeneic tumors were added untreated to the cultures at the
concentrationslisted at the initiation of culture. Tumor agar
colonies were calculated using theplating efficiency of each tumor
(13% for EL4, 27% for J774, and 13% for 70Z/2). PFC estimates are
corrected for tumor cell loads.
Stimu-Mouselatingstrain
tumorC57BL/6
P815BALB/c
EL4BD2F,
C118Increased"
MST:Act-Dara-CMit-CPoly(rl).poly(rC)Restoration0
ofCTL
activity:Act-Dara-CMit-CPoly(rl).poly(rC)Synge-neic
tu-Synge-morcon-neic
tu-centra-mortion/mlEL4
05001,5005,00015,00050,000J774
05001,5005,0001
5,00050.00070Z/2
05001.5005,00015,00050,000Table
8SummarytableEL4—±——+
++——Equivalenttumoragar
colonies0682036752,0256,75001344021,3404,02013.4000722157152,1457,150TumorJ774—±±++
+++
+++%
specific51Cr
re
leased22.627.216.74.31.6035.58.11.700041.238.842.043.733.625.9PFC/1
06 re
coveredcells2792992452431732123825531227924917714551814184718281475151570Z/2+—+—+
+ND+
+++
Restoration ofPFC development:
Act-Dara-CMit-CPoly(rl)-
poly(rC)
ND
+ , greater than 2-fold increase in MST; ±,less than 2-fold
increase in MST;—, no change in MST.
0 + + , complete restoration of suppressed cytotoxic T-cell and
PFC responses; + , partial restoration; ND, not done; —,no
restoration.
response to T-independent antigens was normal. The T-helpercells
required for an antibody response to T-dependent antigens were
markedly reduced in the tumor-bearing mice, suggesting that immune
suppression in tumor-bearing mice mayreside among the T-helper cell
populations.
Claësson and Johnson (3) have reported that B-lymphomaand
mammary carcinoma raise CFU-B and CFU-C levels in thespleens of
tumor-bearing mice. A T-cell lymphoma raised
splenic CFU-B levels but not those of CFU-C. In this study,
noeffect was seen on CFU-B by any of 3 tumors used. In agree
ment with the study of Claësson and Johnson, however, increased
levels of splenic CFU-C were observed in mice bearingthe B-lymphoma
and macrophage tumors (Tables 3 to 6). TheT-lymphoma did not
influence CFU-C. Original to this study isthe influence of tumor on
CFU-M levels. J774 tumors raisedsplenic CFU-M levels in all
experiments. This appears to be
due to a potentiating activity produced by high concentrationsof
the tumor cell line.5 The T-lymphoma did not influence the
CFU-M population, but surprisingly, the 70Z/2 B-lymphoma
markedly depressed the megakaryocyte progenitor levels. Nomarked
decrease in colony size was noted, however.
Drug-induced Restoration and Immune Function. The res
toration of in vitro immune responsiveness in spleen cells
fromtumor-bearing mice was achieved after therapy with
chemicals,each of which affects tumor cells by a different
mechanism(s)(4). Mit-C (Table 3) was very effective in reducing
splenic tumorload in the 3 tumor-host combinations studied.
Restoration ofimmunological responsiveness paralleled tumor
reduction inmice bearing 70Z/2 or J774. This therapeutic effect
resultedin a prolongation of survival in mice bearing 70Z/2.
Totalnucleated cell counts, CFU-B, or PFC responses were
significantly reduced in tumor-free mice receiving Mit-C at
doses
greater than 1 mg/kg/day.The failure of ara-C to overcome the
tumor-dependent sup
pression of immune responses might be related to its lowactivity
against tumor cells established in peripheral organs, asevidenced
by significant tumor load in the spleens at termination of therapy.
Hematopoietic progenitor cell levels and CFU-B-forming capacity in
the spleen were not affected at the dosesadministered in this
study. B-cell responses were severelyimpaired in untreated BALB/c
mice. However, ara-C partiallyrestored immune responses in
tumor-bearing C57BL/6 mice.ara-C has been reported to
preferentially reduce anti-SRBC
responses in dogs receiving renal grafts (12), to reduce
antibody responses to protein antigens in humans (29), and toreduce
serum-blocking factors in tumor-bearing mice (15).
However, it failed to prolong graft rejection (12) and did
notreduce cell-mediated immune responses to mammary tumorantigens
in C3H/HeJ mice (15).
The synthetic double-stranded polynucleotide poly(rl)-poly(rC)
was very effective in reducing tumor load in J774-
bearing mice. This was reflected in a 100% increase in MSTand
complete immunological reconstitution. Mice bearing 70Z/2 tumor
were comparably protected by this agent.6 Doses
higher than 3.0 mg/kg/day appear to be toxic for CTL responses
in normal BALB/c mice, however. C57BL/6 micewere very sensitive to
drug toxicity, in that immune responsiveness was drastically
reduced at levels as low as 0.3 mg/kg/day. This agent differed from
the other drugs tested in thatCFU-C and CFU-M progenitor cell
levels were measurablyenhanced in normal mice receiving
poly(rl)-poly(rC) (Table 5).The elevated progenitor cell levels can
be accounted for bydrug-induced splenic stimulatory factors (26,
42), rather thanmigration, since the regime of 5 mg/kg/day has been
shownto induce marrow aplasia (25).
5N. Williams, unpublished data.eG. Tarnowski, unpublished
data.
4572 CANCER RESEARCH VOL. 39
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-
Therapy of Tumor-induced Splenic Immune Dysfunction
It has previously been reported that the effectiveness
ofpoly(rl)-poly(rC) varies depending on the concentration androute
administered. This agent has been reported to bind totumor cells,
thus enhancing its antigenicity (36). Cellular immune responses
against syngeneic tumors have been reportedto be significantly
increased by pretreating tumor cells with thisdrug. This increase
in antigenicity of the tumor cell couldaccount for increased MST in
J774- or 70Z/2-bearing mice.Immunopotentiation did not occur in the
EL4-C57BL/6 tumor-
host combination since no significant reduction in tumor loadwas
observed. De Clercq ef al. (5) reported that repeateddoses of 5
mg/kg/day showed significant reduction of HT1080fibrosarcoma in
nude mice. This could possibly be due toactivation by
poly(rl)-poly(rC)-induced Interferon of natural
killer cells (7) known to be readily detectable in nude
mice.Although Interferon has been reported to reduce tumor load
inseveral murine systems (13), it did not cause regression of
thehuman HT1080 in nude mice. Levine ef al. (23) were unable
todemonstrate an antitumor response in patients receiving poly(rl)
•poly(rC) therapy for solid tumors or leukemia.
Act-D, an agent active against several human tumors (4, 10),
was only effective in prolonging survival of mice bearing 70Z/2.
Although splenic tumor load was substantially reduced inmice
bearing J774 or EL4, survival was not detectably increased.
Anti-SRBC responses were readily reconstituted to
control levels in each tumor system. CTL responses
wereconcomitantly restored in J774- and EL4-bearing mice. This
agent, administered in dosages of 0.01 mg/kg/day, did
notsignificantly alter total nucleated cell recovery in the spleens
oraffect hematopoietic precursor or CFU-B. Drug-induced aplasia
or preferential infiltration of tumor into bone marrow was
notobserved in treated tumor-bearing animals. We have established7
that ascites cells aspirated from J774-bearing mice
receiving Act-D between 0.003 and 0.01 mg/kg/day are
significantly reduced in their capacity to suppress in vitro
immu-
nological functions compared to untreated ascites cells. The
invitro growth rate and plating efficiency in soft agar were
equalto untreated cells. These results suggest that Act-D affects
the
ability of the tumor to produce factor(s) responsible for
suppression or that the lymphoid cell(s) responsive to the
signalinitiating suppression are effectively depleted by the
drug.Alternatively, Act-D may alter tumor distribution in the
host.
These possibilities are currently being investigated.This study
determined that several tumor cell lines derived
from cells involved in immune responses could
preferentiallyinhibit host immune functions. In certain instances,
the tumor-
induced suppression could be reversed by chemotherapy.Reversal
of immune dysfunction did not always coincide withincreased
survival. Conversely, in no tumor-host drug combi
nation have we observed increased survival without intact
hostimmune responsiveness. Our preliminary data indicate that
asignificant reduction in tumor load (in some cases with
significantly increased MST) in the absence of measurable toxicity
tohematopoietic function and with concomitant restoration ofimmune
responsiveness in tumor-bearing hosts can be
achieved. Such procedures are of importance for the preservation
of immune function in the course of presently usedchemotherapy
regimes and as a basis for developing effective
7 R. Faanes, paper in preparation.
combination schedules which would not impair
immunologicalstatus.
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1979;39:4564-4574. Cancer Res Ronald B. Faanes, Vincent J.
Merluzzi, Neil Williams, et al. T- and B-Cell FunctionsTumor Type
to Prevent Tumor-induced Suppression of Specific Matching of
Chemotherapy to Mouse Strain and Lymphoid
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