Download Effect of Low Dose Cyclophosphamide on the Immune System of

[CANCER RESEARCH 47, 3317-3321, June 15, 1987]
Effect of Low Dose Cyclophosphamide on the Immune System of Cancer Patients:
Reduction of T-Suppressor Function without Depletion of the CD8+ Subset1
David Herd ' and Michael J. Mastrangelo
Division of Oncology, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
ABSTRACT
Low dose cyclophosphamide (CY) can augment the development of
delayed-type hypersensitivity to primary antigens in patients with ad
vanced cancer. In this paper, we have considered the hypothesis that the
immunopotentiation is related to reduction of T-suppressor activity.
Peripheral blood lymphocytes were collected and cryopreserved from 45
patients with metastatic malignancy before and then 3, 7, and 19 days
after administration of CY, 300 mg/mz i.v. The peripheral blood lympho
cytes were tested for generation of concanavalin A-inducible suppressor
activity, proliferative response to phytohemagglutinin, and phenotype
using monoclonal antibodies to ( '1)4 and CDS. Concanavalin A-inducible
suppression was significantly reduced by day 3 and declined progressively
through day 19. The mean percentage changes in suppression were: day
3, -23.4 ±6.8 (SE) (P < 0.01); day 7, -33.1 ±143 (P = 0.052); day
19, -43.1 ±10.7 (P < 0.01). In contrast, CY caused no significant
changes in phytohemagglutinin proliferation (mean percentage changes:
day 3, -4.7 ±6.1; day 7, -15.6 ±7.5; day 19, -5.5 ±8.1), indicating
that the reduction in concanavalin A-inducible suppression was not merely
a reflection of a general reduction in peripheral blood lymphocyte func
tion. The total number of circulating lymphocytes was not affected by
low dose CY. Moreover, flow cytometric analysis showed no significant
changes in the percentage of circulating CD8+ or CD4+ T-cells or in the
CD4/CD8 ratio at any time point after CY. Thus, administration of low
dose CY to these patients caused impairment of nonspecific T-suppressor
function without selective depletion of the CD8+ subset that is generally
associated with that function. Several immunoregulatory models that are
consistent with these observations are discussed.
INTRODUCTION
Cyclophosphamide is a potent immunosuppressive agent that
can also augment a variety of immune responses (1). The major
determinant of whether CY3 suppresses or potentiates immu
nity is the time of its administration in relationship to the
presentation of antigen. Thus, when given following antigen,
CY can suppress the immune response but often augments the
response when given 1-3 days prior to antigen (2).
In experimental systems, CY has been shown to augment
immunity by its selective toxicity to T-lymphocyte-mediated
suppressor function (3). This finding is of more than theoretical
interest, because reduction in T-suppression may be an essential
component of successful immunotherapy, as indicated by sev
eral murine tumor systems (4). For example, Berendt and North
Received 1/2/87; revised 3/20/87; accepted 3/24/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by a grant from the National Cancer Institute (CA39248) and by
funds from the Nat Pincus Endowment.
2To whom requests for reprints should be addressed, at Division of Medical
Oncology, Thomas Jefferson University, 1015 Walnut St., Room 1005, Phila
delphia, PA 19107.
3The abbreviations used are: CY, cyclophosphamide; PBL, peripheral blood
lymphocytes; Con A, concanavalin A; PHA, phytohemagglutinin; 1)111. delayedtype hypersensitivity; MNC, mononuclear cells.
(4) have shown that depletion of T-suppressor cells by CY
markedly facilitates the eradication of an established tumor by
adoptively transferred immune lymphocytes.
For the past several years, we have been attempting to apply
these principles to the treatment of human cancer. We observed
that pretreatment of patients with CY, 300 mg/m2 i.v., 3 days
before injection of antigen augmented the development of de
layed-type hypersensitivity, both to keyhole limpet hemocyanin
(6) and to autologous tumor cells (7). With this publication, we
have begun to investigate the immunological basis of these
findings. The data will show that administration of CY is
followed by reduction in Con A-inducible T-suppressor function
of peripheral blood lymphocytes without a corresponding de
pletion of the suppressor-cytotoxic (CDS-)-) subset.
MATERIALS AND METHODS
Patients. The study population consisted of patients with surgically
incurable, metastatic cancer. Initially, PBL from 13 patients were
studied for Con A-inducible suppression, PHA stimulation, and surface
phenotype. Subsequently, the surface phenotype studies were expanded
by the addition of 32 patients to make a total of 45 study subjects.
A summary of the clinical characteristics of these patients is shown
in Table 1. Thirty-seven of the study subjects had malignant melanoma;
of the other 8, 7 had colorectal carcinoma and 1 had breast adenocarcinoma. Despite the presence of advanced visceral métastasesin most
of these patients, their performance status was good (median Karnovsky
score, 80). Although 34 patients had been treated previously with
chemotherapy and 7 had received radiation therapy, all such cytotoxic
therapy had been discontinued for at least 4 weeks prior to entering
this study. No patients were given corticosteroids while under study.
Treatment. The patients for this study were drawn from two previ
ously published studies: study 1, the effect of low dose CY on the
immunological response to primary, non-tumor-related antigens (6) ( 15
patients); and study 2, the effect of low dose CY on the development
of delayed-type hypersensitivity to autologous tumor cells (7) (30 pa
tients). CY was given at a dose of 300 mg/m2 by rapid i.v. infusion.
Three days after CY, the patients from study 1 were sensitized with
either dinitrochlorobenzene or keyhole limpet hemocyanin, and the
patients on study 2 were given i.d. injections of irradiated, autologous
tumor cells mixed with Bacillus Calmette-Guérin.Heparinized blood
was obtained from all patients just prior to CY infusion (day 0) and
then 3, 7, and 19 days afterwards. No additional treatment was given
during the study period.
Preparation of PBL. As described previously (6), PBL were separated
by density gradient centrifugation on Ficoll:metrizoate, suspended in
freezing medium [consisting of KI'M I 1640 (Grand Island Biological
Co., Grand Island, NY), penicillin, streptomycin, 10% pooled human
AB+ serum (Hint-ell Laboratories, Carson, CA), and 10% dimethyl
sulfoxide], frozen in a controlled rato freezer, and stored in liquid
nitrogen. When needed for testing, MNC were thawed rapidly, diluted
slowly to gradually reduce the concentration of dimethyl sulfoxide, and
washed twice.
Lymphocyte Phenotype Studies. The MNC were stained by incubât
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LOW DOSE CYCLOPHOSPHAMIDE
1Tumor
Table
characteristics of the
patients378291659
typeMelanomaOtherSexMenWomenAgeKarnovsky
statusMétastasesVisceralSkin
(27-79)'80
(40-100)36911142077
onlyPrioror nodal
chemotherapies01>1Prior
AND T-SUPPRESSOR
FUNCTION
calculated the change between the pre-CY sample and each post-CY
sample. Then for a given parameter, we determined whether the mean
change, or percentage change, of the whole group was significantly
different from zero at each time point by using the Student t test for
nonindependent variables (2 tailed). This approach avoids the potential
problem of mistaking spontaneous fluctuations for CY-induced
changes.
RESULTS
Con A-inducible Suppression. The generation of nonspecific
suppressor T-cells was studied in 13 patients before and 3, 7,
therapySurvival
radiation
and 19 days after low dose CY. Suppressor function was signif
(1-50+)
(mo)Clinical
icantly reduced by day 3 and declined progressively through
* Median (range).
day 19. The mean values (percentage of suppression) were: day
0,42.4%; day 3, 33.4%; day 7, 30.4%; day 19, 26.8%. The data
ing 0.5 x IO6 cells with one of the following antibodies on ice for 20
were analyzed by an independent t test (2 tailed) after calculat
min: anti-Leu 3a (anti-CD4; helper-inducer T-cells) (8); anti-Leu 2a
ing the percentage change in suppression at each time point
(anti-CD8; suppressor-cytotoxic T-cells) (8); and anti-Leu M3 (monocompared with the pretreatment (day 0) value (Fig. 1). The
cytes) (9); all were directly conjugated to fluorescein isothiocyanate
mean percentage changes were: day 3, —23.4±6.8 (P < 0.01);
(green fluorescence) or phycoerythrin (red fluorescence) (Becton-Dickday 7, -33.1 ±14.3 (P = 0.052); day 19, -43.1 ±10.7 (P <
inson, Mountainview, CA). Negative fluorescence controls were ob
0.01). The percentage change in suppression on day 19 com
tained by using fluorescein isothiocyanate-conjugated mouse IgG of
pared with day 3 was also significant (mean percentage change,
irrelevant specificity.
-27.3 ±10.8, P < 0.025). A similar degree of impairment of
Analysis of cell surface markers was performed with a Coulter EPICS
Con A-inducible suppression was observed whether patients
C flow cytometer (Coulter Electronics, Hialeah, FL). Lymphocytes
were
sensitized on day 3 with non-tumor antigens or with tumor
were examined by setting bit map gates on a plot of forward versus 90degree light scatter, which excluded monocytes (10); absence of mon - cells (Fig. 1, *, O, respectively).
ocvtes in the bit map was confirmed by lack of staining with anti-Leu
PHA Proliferation. In parallel, PBL from this group of pa
tients were tested for their proliférât
ive response to an optimal
M3.
Blood leukocytes were enumerated in a standard manner with a
concentration of PHA. In contrast to its effect on Con A
Coulter Counter (Coulter Electronics), and differential counts were
suppressors, CY caused no significant changes in PHA reactiv
performed by examination of Giemsa-stained blood smears. Total
ity. The mean responses (cpm) for the group were as follows:
MNC was calculated by adding the proportions of lymphocytes and
day 0, 101, 949; day 3, 98,439; day 7, 104,415; day 19, 89,415.
monocytes, as determined by Giemsa smears, and multiplying the sum
The percentage changes at each time point were analyzed as
by the total blood leukocyte count. The lymphocyte count was deter
described above: day 3, -4.7 ±6.1; day 7, -15.6 ±7.5; day 19,
mined by subtracting monocytes [% Leu M3(+)/100 x total MNC]
-5.5 ±8.1 (P > 0.05 for each) (Fig. 2). Similar results were
from total MNC.
obtained when a suboptimal concentration of PHA (0.1 /¿g/ml)
Con A-inducible Suppression. This was performed by a modification
was used (data not shown). Thus, the progressive reduction in
of the method of Shou et al. (11), which has been described previously
(6). MNC (2 x 10') were incubated in 1 ml of culture medium with or
Con A-inducible suppression seen after CY was not merely a
without Con A (25 ¿ig/ml)in plastic tubes in a 37"C, 5% CU2 incubator
reflection of a general reduction in peripheral blood lymphocyte
for 3 days. Then they were washed and exposed to mitomycin C, 25
function.
¿tg/ml(Sigma Chemical Co., St. Louis, MO), for 30 min in a 37°C
Blood Lymphocyte Composition. Initially, PBL from 10 of
water bath. After 3 washes, they were suspended in culture medium,
and 5 x IO4cells were added to 1 x 10* "indicator" cells in microtiter
wells. The latter were MNC from a normal donor, and the same donor
V40.0-20.0-o.o.-20.
was used in all the experiments described herein. Then PHA was added
to all the wells at a predetermined suboptimal concentration (0.1 ¿¿g/
ml). The mixtures were incubated at 37°Cin 5% COj for 3 days and
Per
then pulsed with ['"Ijiododeoxyuridine (New England Nuclear, Boston,
Cc
MA) and harvested with an automatic harvestor. The percentage of
he
suppression was calculated as
a 0--40.0-60.0-80.0.-100.0.B6M
-p<.01*90M0il
nt
cpm (1 + UN) - cpm (1 + Con A)
ga
100 x
e
cpm (I + UN)
-P-.OKMBH0g
-p<.018N
gPBO.
where cpm (I + UN) are the uptake of ['"Ijiododeoxyuridine
by
indicator cells in the presence of unstimulated patient's cells, and cpm
11
(I + Con A) are the uptake by indicator cells in the presence of Con Astimulated patient's cells.
19
Proliferativi.' Response to PHA. This was performed by a standard
Days After Cyclophosphamide
Fig. 1. Percentage changes in Con A-inducible suppression after cyclophostechnique, described previously (6). The concentration of PHA was 1.0
phamide. Each marker represents the percentage change, compared with day 0,
(jg/ml.
in Con A-inducible suppression generated from the PBL from a single patient. >,
Analysis of Data. Lymphocyte functional assays were performed in
immunized with non-tumor antigens; O, immunized with tumor cells. />, calcu
triplicate and the mean was calculated. Replicate values never varied
lated by a t test for nonindependent samples, has been placed adjacent to the
by more than 20% For each patient and for each parameter, we
mean.
3318
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LOW DOSE CYCLOPHOSPHAMIDE
AND T-SUPPRESSOR
FUNCTION
40.0-20.0-P
Oj40.0h
e.
nnP
"•"c
C
-p-NSN
hSS-»-"'t
•
*if•
M0M3Ä0•*•"ÕNIIMN7
20.0-an9
ga
e -40.0
9e-60.0-80.
ntagBCD60.
nu.
ft
u*-M.O-++
!$
i1
1!!
-|Hß$*
;A.t4+;
0K8Ng
19Days
After
-P-«-•
-P-«
*kt
$
+
CyclophosphamidePee•
Fig. 2. Percentage changes in PHA stimulation after Cyclophosphamide. Each
marker represents the percentage change, compared with day 0, in PHA stimu
lation (cpm) of the PBL from a single patient. *, immunized with non-tumor
antigens; O, immunized with tumor cells. P, calculated by a r test for nonindependent samples, has been placed adjacent to the mean. .V.V.not significant.
-40.0
19
CD8+80.0-,60.0pe
++
the original 13 patients were studied. To reduce the possibility
that small changes in lymphocyte composition would be missed
because of inadequate sample size, PBL that had been obtained
and cryopreserved from an additional 32 patients were studied,
and the results were analyzed compositely. The number of data
points at each time point was: day 0, 42; day 3, 40; day 7, 27;
day 19, 12.
The total number of circulating lymphocytes was not affected
by a single administration of low dose CY; the values (number
of cells per mm3) were: day 0, 1620 ±112; day 3, 1681 ±132;
day 7, 1602 ±152; day 19,1576 ±135. Prior to CY, the T-cell
composition (percentage) measured by flow cytometric analysis,
was: CD4+ T-cells, 46.6 ±2.0; CD8+ T-cells, 19.4 ±1.3; CD4/
CDS ratio, 3.2 ±0.4. Fig. 3 shows the percentage change in
circulating CD8+ and CD4+ T-cells for each of the study
patients following administration of low dose CY. For a partic
ular patient, there was sometimes considerable variation in the
proportion of cells in each subset from one time point to
another. However, analysis of the group demonstrated that
administration of CY was not followed by any significant
changes in circulating CD8+ or CD4+ T-cells or in the CD4/
CD8 ratio, despite a significant change in Con A suppression
and, clinically, an augmented induction of DTH to keyhole
limpet hemocyanin or autologous melanoma cells.
DISCUSSION
The original observation, some 20 years ago, that CY could
potentiate immune responses (12) had been corroborated and
extended by a number of investigators. Most studies have
demonstrated augmentation of cell-mediated immunity, usually
measured as delayed-type hypersensitivity, to an array of anti
gens, varying from microbial products to syngeneic testicular
tissue (13, 14). Under some circumstances antibody responses
have also been heightened (15).
CY-mediated immunopotentiation has been achieved in ex
perimental animals with a remarkably wide range of dosages
from 60 to 2100 mg/m2 (2, 16). However, both in vivo and in
t.
+*•
40.0r
Cc
20.0,e
h
aÃŽS
o.o,a
e9
-ao.O-e-40.
Ã-n
+i
*
+•
i
*
'
^^G-pHC
—TÃ-f^Jr1
±J*
J.
t y
T
-^"«S
+È
+*
*'f
0--60.
i
ÃŽ"
0-+
3
7
19
DaysAfter Cyclophosphamide
Fig. 3. Percentage changes in circulating CD4+ T-cells and CD8+ T-cells
after Cyclophosphamide. Each marker represents the percentage change, com
pared with day 0, for a single patient. /'. calculated by a / test for nonindependent
samples, has been placed adjacent to the mean. .V.V.not significant.
does not require enzymatic activation) at a concentration of 3
Mg/ml, while suppressor T-cells were inactivated at a concentra
tion of 1 Mg/ml or less.
Our laboratory was the first to demonstrate that CY could
augment the human immune response in vivo (17). We showed
that the administration of a conventional cytotoxic dose of CY,
1000 mg/m2, to patients with advanced cancer 3 days before
sensitization with antigen resulted in augmentation of DTH to
the primary antigen, keyhole limpet hemocyanin, with no effect
on the antibody response to that antigen. In a subsequent study
(6), we reported that a much lower dose of CY, 300 mg/m2,
was just as effective in augmenting the acquisition of DTH and
heightened the antibody response as well. More recently, we
have shown that "low dose" CY potentiated the acquisition of
DTH to autologous melanoma cells, which in two cases was
followed by regression of metastatic tumor (7).
In the present study, we have tested the hypothesis that
reduction of nonspecific T-suppressor function might be at least
partially responsible for the immunopotentiating effects of low
dose CY. Using cryopreserved PBL from patients with ad
vitro evidence suggests that lower concentrations of CY may be vanced cancer who had been treated with CY in one of our
advantageous in that they are selectively toxic to the T-supprevious studies, we observed a striking impairment of Con A
pressor system and sparing of other T-cell functions. Thus,
inducible suppressor activity. This reduction was detectable by
Kauffman et ai. (3) reported that T-cells capable of adoptively
the third day after CY administration and increased progres
transferring DTH were resistant to 4-hydroperoxy-CY (which
sively through day 19. The reduction in suppressor function on
3319
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LOW DOSE CYCLOPHOSPHAMIDE
day 3 is consistent with the irnmunopotentiation studies in both
humans and experimental animals (2,6, 7). Thus, sensitization
with antigen on day 3, a time when nonspecific suppressor
function is low, might result in impaired generation of antigenspecific suppression as well, and thus more efficient production
of T-cells that mediate DTH and provide help for antibody
production. The persistence of the impairment of Con A sup
pression through day 19 is more difficult to correlate, since in
the few human studies and in most of the published animal
experiments the interval between CY and immunization has
been no more than 3 days. However, at least one laboratory
(18) has shown that immunopotentiation can be obtained in
mice when antigen is given as long as 7 days after CY, and in
humans the potentiation window may be considerably wider.
Since Con A-inducible suppression is generally associated
with the CD8+ subset of T-cells (19), it is noteworthy that the
impairment ofl -suppressor activity was associated neither with
a fall in the percentage of circulating CD8+ cells nor with a
change in the so-called "helper/suppressor" ratio, i.e., %CD4+/
%CD8+. Previously, we found that high dose (1000 mg/m2)
CY inhibited Con A-inducible suppression without changing
the relative proportions of CD4+ and CD8+ T-cells, but in
that study, there was a marked reduction in the total blood
lymphocyte concentration and thus a fall in the absolute number
of CD8+ T-cells (20). In contrast, in the current study low dose
CY reduced suppressor function without affecting either the
relative or the absolute numbers of this subset.
These results are in conflict with data published by Bast et
al. (21) who reported that low doses of CY (200-400 mg/m2)
did produce selective depletion of circulating CD8+ T-lymphocytes in patients with melanoma. However, their conclusion
was based on the study of only 4 patients, 3 of whom exhibited
a significant change. Moreover, they administered multiple,
escalating doses of CY to each patient and thus could have been
measuring a cumulative effect rather than the effect of a single
dose. We believe that our study (testing a single administration
of CY, using a much larger sample size, and performing statis
tical analysis by group) provides a more accurate assessment of
the effects of low dose CY on peripheral blood T-cell compo
sition.
There are several immunoregulatory models that are consist
ent with the observation that CY reduces Con A-inducible
suppression without depleting CD8+ T-cells. Ozer et al. (22)
have shown that in vitro exposure of human lymphocytes to 4hydroperoxy-CY prevents the generation of suppressor T-cells
by Con A but does not inhibit the suppressor cells after they
have been induced. They postulated the existence of a minor,
extremely CY-sensitive, population of "presuppressor" cells.
Secondly, Damle et al. (23) have studied a T-cell with the
phenotype CD4+, Leu 8+, which is not a suppressor but is
necessary for the induction of CD8+ suppressors. Finally,
evidence has accumulated that CD4+ T-cell themselves can
become potent suppressors after stimulation with Con A (24).
Morimoto et al. (25) have suggested such cells bear the newly
recognized surface molecule, 2H4. Partial depletion of, or sublethal damage to, one of these subpopulations of CDS- lym
phocytes by CY could be reflected in reduction of T-suppressor
activity and, consequently, in augmentation of immune re
sponses.
AND T-SUPPRESSOR
FUNCTION
ACKNOWLEDGMENTS
The authors wish to thank the following individuals for their inval
uable assistance in the completion of this project: Ellen Hart, R.N.,
who performed the patient-related work; Marsha Golden, who per
formed the flow cytometry; and Cannella Clark, who performed the
lymphocyte functional assays.
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LOW DOSE CYCLOPHOSPHAMIDE
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Effect of Low Dose Cyclophosphamide on the Immune System
of Cancer Patients: Reduction of T-Suppressor Function without
Depletion of the CD8+ Subset
David Berd and Michael J. Mastrangelo
Cancer Res 1987;47:3317-3321.
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1987 American Association for Cancer Research.