Cancer Epidemiology Biomarkers & Prevention Vol. 9, 609-617, June 2000
© 2000 American Association for Cancer Research
Blood Level of B and CD4+ Lymphocytes Measured before Induction of an Experimental Tumor in Rats Predicts Tumor Progression and Survival1
Melissa K. Demetrikopoulos2,
Ronald H. Goldfarb,
Zhao-bin Zhang and
Jay M. Weiss3
Emory University School of Medicine, Department of Psychiatry and Behavioral Sciences, Atlanta, Georgia 30322 [M. K. D., Z-b. Z., J. M. W.], and University of North Texas, Health Science Center at Fort Worth, Department of Molecular Biology & Immunology, Fort Worth, Texas 76107 [R. H. G.]
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Abstract
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After an initial series of experiments indicated that early responses of
B lymphocytes were important in controlling tumor metastases in two rat
models of cancer (N. Quan et al., Cancer Res.,
59: 1080–1089, 1999), the present study assessed
whether differences in the number of B lymphocytes that are normally
present in different individual rats before any tumor development could
predict tumor growth, metastases, and length of survival when tumor
challenge subsequently occurred. Repeated baseline measures of several
circulating lymphocyte subtypes (i.e., natural killer,
B, CD4+, CD8+ lymphocytes) were made in individual inbred WAG
rats before any introduction of tumor cells, and stable baselines for
these subtypes were found. Animals were then injected with 2 x
106 CC531 tumor cells (a syngeneic tumor) into the leg, and
the size of the resulting primary tumor measured. Primary tumors were
surgically removed 6–7 weeks after tumor-cell injection, and animals
then followed until death from metastases. In two experiments, the size
of the primary tumor as well as the length of time that animals
survived correlated with the pretumor percentage of certain lymphocyte
subtypes in peripheral blood before tumor-cell injection. Baseline
percentage of B lymphocytes was significantly negatively correlated
with the size of the primary tumor and was positively correlated with
the duration of survival. Baseline percentage of CD4+ lymphocytes
showed the opposite relationship, being positively correlated with
tumor size and negatively correlated with survival time, although these
correlations were lower than those for B lymphocytes. Percent B
lymphocytes in circulation also declined during tumor development. In
summary, a high percentage of endogenous peripheral blood B lymphocytes
predicted growth of smaller primary tumors and longer survival after
experimental tumor induction in a rat model, further suggesting that B
lymphocytes are involved in protection against development of certain
tumors.
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Introduction
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Although development of various experimental tumors is apparently
controlled by
NK4
cells [e.g., the studies of Gorelik et al.
(1)
and Barlozzari et al. (2)
],
we recently found evidence pointing to the direct involvement of B
lymphocytes in the control of some of these tumors (3)
. In
this paper, we reported that the most marked change in a lymphocyte
subtype that occurred in different compartments after i.v. injection of
tumor cells that metastasize to lung was a rapid accumulation of B
lymphocytes in the lung. Two further manipulations then suggested that
B lymphocytes were involved in the control of tumor development. First,
immunoneutralization of B lymphocytes by injection of a high dose of
antibody to rat B lymphocytes increased development of lung tumors
after i.v. tumor-cell administration. Second, inoculation of the
Fischer 344 rats with MADB106 tumor given into the leg, which makes
these rats resistant to development of lung tumors when MADB106 is
subsequently given i.v., resulted in an increased level of B
lymphocytes in the lung of these "lung-tumor resistant" animals.
Experimental studies from other laboratories have also implicated B
lymphocytes in the control of tumor growth and metastasis. Yuhas
(4)
found that infusion of splenic B lymphocytes into
BALB/c mice reversed radiation-induced enhancement of alveolar cell
carcinoma metastases. Guo et al. (5)
reported
that injection of cells produced by the fusion of activated B
lymphocytes with hepatocellular carcinoma cells protected against
subsequent tumor challenge and had antitumor activity against
established tumors in Wistar rats. Also, effects of antitumor agents in
nude versus SCID mice point to the importance of B
lymphocytes in tumor surveillance. Because SCID mice lack both B and T
lymphocytes, whereas nude mice only lack T lymphocytes, differential
responses of these strains have been used to assess the contribution of
B lymphocytes in tumor-related phenomena. Quin and Blankenstein
(6)
found that lymphotoxin produced by transfected J588L
cells led to tumor rejection in syngeneic BALB/c mice, partial
rejection in nude mice, and lack of rejection in SCID mice. The
differential antitumor effect of lymphotoxin in nude and SCID mice
suggests the involvement of B lymphocytes in antitumor action of
lymphotoxin. Similarly, Reisfeld et al. (7)
reported that an antibody-lymphotoxin fusion protein inhibited
metastases in athymic nude mice but not in SCID mice.
The experiments described in this paper attempted to extend our earlier
findings. Whereas we previously used immunoneutralization to manipulate
B lymphocytes (3)
, the present study examined whether
naturally occurring individual differences in B lymphocytes might be
related to tumor development and survival. In these studies, inbred
WAG rats were injected with syngeneic CC531 tumor cells into the
ventral aspect of the hind leg so they would develop a primary tumor at
this site. Before tumor cells were injected, blood samples were taken
from each rat, and the percentage of various lymphocyte subtypes (NK,
B, CD4+, and CD8+) were measured so that a pretumor circulating level
of these lymphocyte subtypes was determined for each animal. After
tumor cells were injected, the size of the primary tumor was measured
at various intervals for 6–7 weeks, at which time the primary tumor
was removed and the length of time that animals survived was measured.
The intent of the study was to determine if naturally occurring
differences in the percentage of any blood lymphocyte subtype present
before tumor cells were injected would prove to be related to
(i.e., correlated with) the development of the primary tumor
(i.e., size of tumor), development of metastases, and
duration of survival.
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Materials and Methods
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Animals.
Female inbred WAG (Harlan/CPB, Zeist, the Netherlands) rats >8 months
of age were used. Animals were housed four to a cage in microisolator
cages under positive-pressure ventilation, with cages maintained on
closed-shelf, laminar-flow racks. Thus, rats in any given cage were not
in contact with pathogens, odors, or noises of other animals in the
colony. The animal colony room was maintained at 22°C ± 1°C
on a 12/12 light/dark cycle with food and water available ad
libitum. All procedures involving the use of animals were approved
by the Institutional Animal Care and Use Committee.
Blood Sampling Procedure.
Samples of peripheral blood were collected in heparinized Natelson
tubes by nipping the end of the subjects’ tail with a sterile scalpel
and gentle milking of the tail. Blood samples were collected beginning
3.0 h after lights-on in the colony. For sampling, a cage containing
four animals was removed from the rack, and each animal was taken
individually into an adjoining room where the blood sample was taken.
After taking of the blood sample, each animal from a cage was placed
into a holding cage in the adjoining room until all four animals of the
cage had been sampled, at which time the four animals were returned to
their home cage and the cage was replaced into the rack. Completing
blood sampling for a cage required 8–10 min. Blood was drawn from the
animals of a cage in the same order on each occasion when sampling was
done; subsequent analysis determined that the percent of lymphocyte
subtypes found in blood was unrelated to the order of blood sampling of
the animals in a cage. Completing blood sampling from all animals in a
study required 1 h.
Percent Lymphocyte Determination.
Two samples of 200 µl each were taken for florescent labeling of
lymphocytes and the quantification of lymphocyte subtypes [NK, B
(CD45RA), CD4+, CD8+] on a flow cytometer using standard techniques.
Details of the procedure used were given previously in Quan et
al. (3)
. Briefly, each sample was incubated for 45
min at 4°C after the addition of 50 ml of 1:200 dilution of
monoclonal antibodies to FITC-conjugated antiCD4 (OX-38) and
R-phycoerythrin-conjugated antiCD8 (OX-8) to one sample and
R-phycoerythrin-conjugated antiCD45RA (OX-33) and 1:300 dilution of
FITC-conjugated anti-NK (NK 3.2.3) to the other sample. (Dilutions used
were determined by serial dilution to give the best staining of the
above lymphocyte surface markers.) Samples were analyzed on a
fluorescence-activated cell sorting CALIBER (Becton Dickinson)
cytometer. Positive staining was gated by subtracting autofluorescence
from unstained control samples.
Tumor Cell Culture.
The syngeneic CC531 cell line, derived from a
1,2-dimethylhydrazine-induced adenocarcinoma of the colon
of a WAG rat (8)
, was used (generously supplied by Dr.
Peter Kuppen [Leiden, the Netherlands]). Cell lines were
maintained in 5% CO2 at 37°C in monolayer
cultures in complete media (RPMI 1640) supplemented with 10%
heat-inactivated fetal bovine serum, 0.01 mg/ml gentamicin, 2
mM L-glutamine, 0.1 mM nonessential
amino acids, and 1 mM sodium pyruvate. Cultures were
harvested with 0.25% EDTA in HBSS, washed, and suspended in
Dulbecco’s PBS [8 g/liter NaCl, 1.15 g/liter
Na2HPO2 (PBS)] before
injecting into animals. Cell cultures used had undergone <10 passages.
Induction of Tumor, Measurement of Primary Tumor Size, and
Assessment of Tumor Metastases.
Rats were lightly anesthetized with halothane, and 2 x
106 tumor cells in 1 ml of PBS were injected s.c.
on the ventral surface into the upper thigh region of the left leg. The
size of the primary tumor that developed at this site was determined by
lightly anesthetizing the animal, palpating the tumor to determine its
extent, and then measuring the tumor with microcalipers. The diameter
was calculated by averaging the length and width measurements, which
gave an accurate representation of the tumor size as evidenced by tumor
diameter being correlated r = +.94 with tumor weight in
grams in experiment 1 and r = +.90 in experiment 2. For
removal of the primary tumor, animals were anesthetized with halothane,
an incision was made in the leg skin to expose the s.c. tumor, and the
tumor was dissected out. The wound was then closed with surgical
staples, and following a brief recovery period, the animal was returned
to its home cage. Animals remained in the home cage until death
appeared imminent (indicated by weight loss, decrease in grooming and
mobility, and difficulty breathing) at which time the animals were
sacrificed by opening of the chest cavity under halothane anesthesia.
At this time, animals were necropsied for presence of metastases
throughout the body. Lung metastasis was quantified using the method
described in Wexler (9)
. For this procedure, after opening
of the chest cavity, the lungs were infused with 15% India ink through
the trachea, which causes the lung surface to stain black except for
tumors that resist dye and thus are white. The number of metastasis on
the lung surface was subsequently counted.
Statistical Analysis.
Correlations reported are Pearson Product Moment correlations.
Comparisons between groups were done by t tests.
Procedure of Experiment 1.
Four blood samples (with a 2-week interval between each sample) were
taken before tumor-cell injection and analyzed for composition of
lymphocyte subtypes. Sixteen animals were used. After collection of the
fourth sample from all animals, each animal was anesthetized as
described, and tumor cells were injected. On days 21 and 47 after
tumor-cell injection, a blood sample was taken after which the size of
the primary (leg) tumor was measured. On day 49, the primary tumor was
surgically removed. Thereafter, animals remained undisturbed in the
home cage until death appeared imminent, at which time the animal was
removed and sacrificed, and a necropsy was conducted to assess
metastases throughout the body.
Procedure of Experiment 2.
The procedure of this experiment was the same as in experiment 1 except
that the primary tumor was removed earlier than in the previous
experiment (day 41 after tumor-cell injection) to reduce the
possibility of any mortality arising directly from the primary tumor
and thus increase longevity in the experiment. Sixteen animals were
used. In addition, three other animals were treated exactly as the 16
animals described above, including surgery on day 41, but these were
injected with vehicle containing no tumor cells. These animals given no
tumor cells were included to determine whether changes in blood
lymphocyte subtypes that occurred after tumor-cell injection were
attributable to the presence of tumor or occurred simply because of
repeated sampling or other manipulations. As in experiment 1, animals
remained undisturbed in their home cage after tumor-removal surgery
until death appeared imminent, at which time the animal was sacrificed,
and a necropsy was conducted.
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Results
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Baseline Measurement of Lymphocyte Subsets.
Table 1
shows, for each of the lymphocyte subtypes that was measured, the
correlation between each of the four blood samples taken before tumor
cells were injected. In both experiments, all of these correlation
coefficients were statistically significant (P < .05)
except one. That these correlation coefficients were significant
indicates that the composition of blood lymphocyte subtypes found in
individual animals was similar from one sampling to another, and thus
individual animals show statistically significant stability with
respect to these subtypes of circulating lymphocytes. For each
lymphocyte subtype, the average of the four measures made before
tumor-cell injection was computed for each animal, and this constituted
each animal’s "baseline" value for that lymphocyte subtype.
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Table 1 Correlations between percent of different lymphocyte subtypes (NK, B,
CD4+, and CD8+) measured in blood samples taken
at 2-week intervalsa
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Growth of Primary Tumor.
On the day that the fourth blood sample was taken, tumor cells were
injected into the leg, after which, as expected, a primary tumor
subsequently developed at the site of the injection. In experiment 1,
the average tumor size on day 21 after injection was 20.2 (±1.6) mm in
diameter, and this progressed to 34.4 (±1.7) mm by day 47. The
validity of the measurement technique used to measure the size of the
primary tumor in vivo was confirmed when the tumor was
removed on day 49, weighed, and the weight of the excised tumor
correlated with the measurements of size. The average tumor weight on
day 49 was 16.6 (±2.0) g, and, more important, the weight of the
individual tumors correlated r = +.93 with measurement
of diameter made on day 47. In experiment 2, smaller primary tumors
developed than were seen in experiment 1. On day 21 after injection,
average tumor size was 13.4 (±1.5) mm, and this progressed to 20.5
(±4.6) mm on day 41. The weight of tumor when it was removed on day 41
again correlated highly (r = +.90) with the size
(diameter) measured in vivo on that day. It can be noted
that in experiment 2, considerable variability of tumor size was seen
on day 41 because in this study, some tumors actually became
smaller than they had been on day 21—whereas in experiment 1 every
animal showed a larger primary tumor on the second measurement than the
first, six animals in experiment 2 showed a decrease in primary tumor
size on the second measurement. Of these, two animals in experiment 2
apparently had resolved the primary completely by the second
measurement; their primary tumor, which had been of measurable mass on
day 21, was no longer evident on day 41 either by palpation or on
inspection of the region when it was opened surgically for tumor
removal.
Relationship of Lymphocyte Subtypes in Blood before Injection of
Tumor Cells to Size of Primary Tumor.
The top six rows in Table 2
show correlations between the baseline value of each lymphocyte subtype
(i.e., the average value computed from the four blood
samples taken before injection of tumor cells) and the size of the
primary tumor measured in each animal in vivo on day 21 and
on day 47 (experiment 1) or 41 (experiment 2) after tumor-cell
injection, as well as the weight of this tumor when it was removed. In
both experiments 1 and 2, a significant relationship was seen between
the baseline value for B lymphocytes and the size of the tumor on day
21 after tumor-cell injection; these correlations were the largest
observed in the study. These relationships were negative, indicating
that a high percentage of B lymphocytes at baseline was associated with
small tumor size. The baseline value of B lymphocytes was also
significantly correlated with tumor size on days 47 or 41 and weight of
tumor when it was removed, although these correlations were not as
large as those with tumor size on day 21. The other lymphocyte subtype
whose baseline value was related to tumor size was CD4+ cells. The
percent of these lymphocytes measured before tumor-cell injection was
positively related to tumor size, meaning that a high percentage of
CD4+ lymphocytes was associated with large tumor size. Interestingly,
the baseline value for NK cells showed no relationship to subsequent
tumor size despite the fact that growth of the CC531 tumor has been
found to be under NK surveillance. Baseline values of CD8+ cells also
were not significantly related to tumor size (except in one instance).
Percentages of lymphocyte subtypes in circulation on the days that
tumor size was measured were also correlated with the size of the tumor
measured on those days, and these correlations are shown in the lower
four rows of Table 2
. On days when tumor was measured, the percent of B
and CD4+ lymphocytes in circulation on those days had a significant
relationship to tumor size, whereas NK and CD8+ lymphocytes also showed
this relationship but only in the first experiment.
Relationship of Lymphocyte Subtypes in Blood before Injection of
Tumor Cells to Duration of Survival.
Table 3
shows the frequency and location of metastases found at the necropsy of
the animals in experiments 1 and 2. All animals that were necropsied,
which was done when an animal approached death, had extensive
metastases in lung, often >1000 colonies in the lung, and this was the
apparent cause of death in all cases. Metastases were also found in
lymph nodes, colon, and thymus. Four animals in experiment 2 lived
beyond the 6-month cutoff for the study and consequently metastases
were not assessed in these animals.
An important focus of the study was to determine whether baseline
measures of lymphocyte subtype (i.e., pretumor measures)
might be related to the length of time that animals survived when they
were subsequently injected with tumor cells. To assess this
relationship in experiment 1, the animals were divided into groups
representing the extremes of survival. In experiment 1, no animal
survived beyond 116 days after tumor cells were injected. Six rats
survived the longest, all of these living >90 days after tumor-cell
injection; these were designated as "long survivors." Five rats in
this study formed a group that survived for the least duration, all of
these living for <65 days after tumor-cell injection; these were
designated as "short survivors." Fig. 1
shows average baseline lymphocyte values of the long and short
survivors. Whereas no difference for any subtype reached statistical
significance, the baseline value of B lymphocytes showed the largest
difference between long and short survivors, with long survivors
tending to have higher baseline B lymphocytes than did short survivors.
This difference approached, but did not reach, statistical
significance.
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Fig. 1. Baseline percent of B, NK, CD4+, and CD8+ lymphocytes
(i.e., average from four blood samples taken at 2-week
intervals before tumor-cell injection) in short and long survivors in
experiment 1. Short survivors (n = 5) lived for
<65 days after tumor-cell injection, whereas long survivors
(n = 6) lived >90 days after tumor-cell injection;
these groups represent the extremes of survival duration in experiment
1 (see far right panel). Means and SEs are presented.
*, differs from short survivors at P <
.09.
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As pointed out in the procedure, in experiment 2 the primary tumor was
removed earlier than in experiment 1 (i.e., 41 days after
tumor cells were injected versus 49 days) to attempt to
increase differences in longevity between individual animals and thus
permit better evaluation of any relationship between lymphocyte
baseline values and duration of survival. Baseline values for two
lymphocyte subtypes, B and CD4+, were found to be related to survival
duration in experiment 2. Fig. 2
shows the relationship between the baseline value for B lymphocytes and
survival duration for each animal in the study, and Fig. 3
shows the same relationship for CD4+ lymphocytes. As Fig. 2
shows, high
baseline values of B lymphocytes were associated with long survival; in
fact, those animals with the highest pretumor levels of B lymphocytes
were still alive 180 days (6 months) after tumor-cell injection. The
correlation of baseline B-lymphocyte value and number of days of
survival shown in Fig. 1
was r = +.63
(P < .05). Survival duration was also related to
baseline CD4+ lymphocytes, which is shown in Fig. 3
. The baseline value
of CD4+ lymphocytes was negatively related to survival—animals with
low CD4+ lymphocytes before tumor-cell injection lived longer than
animals with high CD4+ lymphocytes. The correlation of baseline CD4+
lymphocyte values and number of days of survival was r = -.58 (P < .05). Not significantly related to
survival were baseline values of NK cells (r = -.04)
or CD8+ cells (r = +.30).
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Fig. 2. Baseline percent of rat B lymphocytes (CD45RA+ lymphocytes) in blood
(i.e., average from four blood samples taken at 2-week
intervals) of 16 WAG rats shown in relation to the length of time that
these rats survived after being injected with 2 x 106
CC531 tumor cells into the leg. The primary tumor was removed
surgically on day 41.
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Fig. 3. Baseline percent of rat CD4+ lymphocytes in blood (i.e.,
average from four blood samples taken at 2-week intervals) of 16 WAG
rats shown in relation to these length of time that the rats survived
after injected with 2 x 106 CC531 tumor cells into
the leg. The primary tumor was removed surgically on day 41.
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Changes in Circulating Lymphocyte Subtypes during Tumor
Development.
Because blood samples were taken and lymphocyte subtypes were measured
on the days that tumor size was determined, changes in circulating
lymphocyte subtypes occurring in the course of tumor development also
could be assessed. Fig. 4
shows the percentage of lymphocyte subtypes on the days that tumor size
was measured in both experiments 1 and 2; baseline values are also
shown to permit evaluation of change as tumor developed. Considering
that changes in blood lymphocyte subtypes might vary depending on rate
of tumor growth and duration of survival, these data are presented for
long and short survivors—for experiment 1, these groups are defined as
described in the previous section, whereas for experiment 2, six
animals survived >100 days (long survivors) and the remaining 10
animals survived no more than 80 days (short survivors; see Fig. 2
).
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Fig. 4. Percent of lymphocyte subtypes (B, NK, CD4+, CD8+) in blood at baseline
(i.e., average of four blood samples taken at 2-week
intervals before tumor-cell injection) and on days 21 and 47
(experiment 1) or 41 (experiment 2) after tumor-cell injection in short
and long survivors in both experiments 1 and 2. See
"Results" for the definition of short and long survivors.
Experiment 2 also included rats measured at each time point but not
injected with any tumor. Also shown (bottom) is the size
of the primary tumor. Means and SEs are presented. *, differs
significantly (at least P < .05) from baseline (by
dependent t test comparing each animal’s level with its
own baseline value).
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In experiment 1, similar changes occurred in both long and short
survivors, although some changes were more pronounced in the short
survivors than in the long survivors. The percentage of B and CD8+
lymphocytes markedly decreased as tumors developed. NK cells were also
decreased in tumor-bearing animals relative to baseline, but the
decrease was not progressive as the percentage of these lymphocytes
increased somewhat on day 47. CD4+ lymphocytes, in contrast to the
other lymphocyte subtypes, showed a marked increase with tumor
progression. In experiment 2, a small number of additional animals that
were not injected with tumor (n = 3) were included in
the study to assess the possibility that changes seen in lymphocyte
percentage might be simply attributable to repeated sampling or assay
variability. Changes in circulating lymphocytes during tumor
development were considerably less pronounced in experiment 2 than
in experiment 1. The only change seen in tumor-bearing animals in
experiment 2 that was (a) similar to what was seen in
experiment 1, and (b) also did not occur in "no tumor"
animals and thus could be attributed to progression of tumor was a
decline in circulating B lymphocytes—a highly significant fall from
baseline occurred in circulating B-lymphocyte percent in short
survivors, with much less decline occurring in long survivors (whose
average primary tumor size actually decreased on the second measurement
relative to the first; see lowest panel of Fig. 4
).
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Discussion
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The results described in this paper showed that the percentage of
certain lymphocyte subtypes measured in blood before the induction of
an experimental tumor predicted the growth (size) of the primary tumor
as well as the development of metastases and duration that animals
survived after the tumor-cell injection. The present study was
stimulated by our earlier results, which suggested that B lymphocytes
exert an inhibitory influence over tumor development in certain animal
models (3)
. Consistent with these earlier findings,
differences between individual rats in the percentage of endogenous
circulating B lymphocytes showed a significant negative relationship to
the size of a CC531 primary tumor that WAG rats developed when tumor
cells were injected as well as to the time of death from metastases.
Thus, animals with the highest percentages of circulating B lymphocytes
before tumor-cell injection developed the smallest primary tumors and
lived longest after tumor cells had been injected. Even across the two
experiments that were conducted, results consistent with this
relationship appeared— the animals in experiment 1 had significantly
lower baseline levels of circulating B lymphocytes than did the animals
in experiment 2, and the animals in experiment 1 developed larger
primary tumors than did the animals in experiment 2 and also died
earlier. The results described in this paper contribute further
evidence suggesting that B lymphocytes affect certain experimental
tumors by showing the importance of endogenous B lymphocytes, thereby
adding to previously reported findings that had shown this phenomenon
by using experimental manipulation of B lymphocytes (i.e.,
immunoneutralization) to affect tumor growth.
It can be noted that the relationship between B lymphocytes and tumor
development reported in this paper may not be unique to the WAG
rat-CC531 tumor model. In a previous study, we injected Fischer 344
rats s.c. with MADB106 tumor cells to produce a primary tumor of this
type (3)
. In that study, one of the animals completely
resolved its s.c. MADB106 tumor. When B lymphocytes in lung were
assessed (only lung was measured in this study), this animal was found
to have by far the highest percentage of B lymphocytes of any animal in
the study.
The mechanism through which B lymphocytes influence tumor development,
however, is not yet evident from these studies. Of considerable
importance, the earlier study (3)
showed that
immunoneutralization of B lymphocytes had to occur in very close time
proximity to the introduction of tumor cells to augment tumor
development. Delaying administration of the neutralizing antibody to B
lymphocytes by even 1 or 2 h after the tumor cells were injected
resulted in significant and time-related decreases in the ability of
immunoneutralization to increase tumor development, with a delay of
24 h resulting in no effect of the immunoneutralization. This
finding indicates that antitumor effects of B lymphocytes depend on
events that occur very soon after tumor cells are present at the site
of tumor progression. In that any production of antibodies to the tumor
by B lymphocytes would occur at moderately long time intervals after
the introduction of tumor antigen (i.e., days), the observed
time course of the effects of immunoneutralization indicates that
antitumor actions of B lymphocytes are mediated, at least in part, by
functions of B lymphocytes other than production of antitumor
antibodies. Various other functions of B lymphocytes can be suggested
to underlie their antitumor activity. First, B lymphocytes might
enhance cytotoxicity of NK cells, either by increasing production of
IFN-
by NK cells (10)
or by themselves releasing
interleukin 12, which markedly potentiates NK cell cytotoxicity
(11)
. Second, antitumor activity of B lymphocytes may
derive from their functioning as antigen-presenting cells, in that
antigen presentation is important in tumor killing (12
, 13)
. B lymphocytes have been found to bind, internalize, and
present antigens [reviewed by Pernis and Weber (14)
].
CD4+ lymphocytes also were related to tumor growth, but this
lymphocyte subtype showed the opposite relationship from that shown by
B lymphocytes, i.e., baseline CD4+ lymphocyte percentage was
positively related to primary tumor size and negatively related to
length of survival. Thus, animals with high percentages of CD4+
lymphocytes before tumor-cell injection developed the largest primary
tumors and died soonest after being injected with the tumor. An obvious
question is whether the reciprocal relationship between B and CD4+
lymphocytes is an artifact of the measure used, which was the
percentage of total lymphocytes. It can be asked whether a high
percentage of CD4+ lymphocytes might appear to be related to tumor
growth simply because this measure would necessarily rise in any
instance where the percentage of B lymphocytes is reduced, or
vice versa. However, analysis of the data shows that this is
not the case, but that both B and CD4+ lymphocytes make a separate
significant contribution to predicting tumor growth and mortality. For
example, in experiment 1, baseline percent B lymphocytes correlated
-.63 with tumor size of day 21, and baseline percent CD4+ lymphocytes
correlated +.51. Calculation of a multiple correlation using the
explanatory value of both B and CD4+ baselines yielded a multiple
r of .88, which meant that using both B and CD4+ baselines
explained 77% of the variance in tumor size on day 21, whereas the B
lymphocyte baseline alone explained 40% of the variance. Thus, adding
baseline percent of CD4+ cells increased the amount of variance that
was explained by the largest of the individual correlations
(i.e., that of baseline percent B lymphocyte) by 37%, which
is a highly statistically significant addition (P <
.01). That percent of B and of CD4+ lymphocytes each made a
statistically significant independent contribution to predicting tumor
size shows that the two measures do not owe their ability to predict
tumor size to their being correlated with one another.
It was also observed that the percentage of B lymphocytes in
circulation declined as the experimental tumor grew. A fall in
circulating NK and CD8+ lymphocytes was seen in experiment 1, but only
the fall in B lymphocytes appeared consistently in both experiments. A
possible explanation for why this occurs is that B lymphocytes move out
of circulation as part of the host response to control tumor
development and that B lymphocytes then accumulate in tumor sites
and/or lymph nodes during the growth process because these lymphocytes
are directly involved with tumor surveillance at the site of tumor
cells. This possibility would be consistent with our previous findings
(3)
reporting accumulation of B lymphocytes in lung when
tumors that metastasize in lung were introduced. It also would explain
why animals having a high percentage of circulating B lymphocytes
develop smaller tumors, reduced metastases, and survive longer, in that
such animals would have large numbers of circulating B lymphocytes
available for combating tumor development.
The results described here relate to previous clinical findings
regarding B lymphocytes in cancer development. In the 1970s, studies
examined lymphocyte number and subtype composition in peripheral blood
of cancer patients, and several of these studies reported that a
reduced level of B lymphocytes in the peripheral blood of cancer
patients was prognostic of poor outcome [reviewed in Lee
(15)
]. Similar findings were subsequently reported by
Domagala et al. (16)
and Wood and Neff
(17)
, the latter investigators arguing that the decrease
in B lymphocytes could not be accounted for by monocytosis in the
cancer patients. Recently, Kay et al. (18)
reported that patients with multiple myeloma showed low circulating
levels of CD19+ B lymphocytes in clinical stage III and that high
circulating levels of CD19+ B lymphocytes were associated with longer
event-free survival and clinical response. Similarly, Rawstron et
al. (19)
found a suppression of CD19+ B lymphocytes
in presentation or relapse stages of multiple myeloma. The finding
reported in this paper that circulating B lymphocytes declined in the
WAG rat during tumor development would appear consistent with the
findings reported in these papers. However, it should be noted that not
all investigations have observed a decrease in circulating B
lymphocytes during cancer development because Stein et al.
(20)
reported elevated levels of circulating CD5+
lymphocytes in cancer patients with a variety of tumors. On the other
hand, this last finding might not be contradictory but instead indicate
that different subpopulations of B lymphocytes are affected differently
during cancer development. It should be noted that these clinical
findings were obtained in patients who already had cancer, whereas the
present study is prospective in design and demonstrates for the first
time that lymphocyte subtypes in peripheral blood before cancer
development are predictive of survival after tumor challenge.
Further research is needed in animal models to define the nature of the
role that the B lymphocyte plays in the progression of some tumors and
in human populations to determine if monitoring of lymphocyte subtypes
is clinically relevant for predicting tumor development and metastases.
|
Acknowledgments
|
|---|
CC531 cells were a generous gift from Dr. Peter Kuppen (Leiden,
the Netherlands). We thank Robert Donahoe for use of his
fluorescence-activated cell sorting machine and Lorna Clarke and Sandra
Parks for their assistance in preparation of the manuscript.
|
Footnotes
|
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by the Brain, Behavior and Immunity
project (Bruce McEwen, Director) of the John D. and Catherine T.
MacArthur Foundation; and National Institute of Mental Health
Individual National Research Service Award MH10952 (to M. K. D). 
2 Present address: Department of Biology, Georgia
State University, Atlanta, GA 30303.
3 To whom requests for reprints should be
addressed, at Emory West Campus, 1256 Briarcliff Road, NE, Atlanta, GA
30306. Phone: (404) 712-9772; Fax: (404) 712-9755; E-mail: jweis01{at}emory.edu
4 The abbreviation used is: NK, natural killer.
Received 11/11/99;
revised 3/15/00;
accepted 3/28/00.
|
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Abstract
Introduction
