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Department of Surgery, University of California, San Francisco, VA Medical Center at San Francisco, San Francisco, CA 94121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. The contribution of Cy is
probably due to immunopotentiation of DTH rather than to direct
cytotoxicity to the tumor. The regression of these large tumors takes
>4 wk and, in many cases, is self-sustained, in that little or no
additional IL-12 is needed beyond the initial week of administration.
Analysis of the cellular and molecular events at the tumor site
suggests that the mechanism is a Th1-mediated antitumor immune
response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by NK (7) and
T cells (8), and CTL development in vitro (9). It is also found to
possess antiangiogenesis activity, apparently through the induction of
IFN--inducible protein 10
(IP-10)2 (10). Until
recently, IL-12 was thought to contribute to antimicrobial responses by
activating the production of IFN- by T or NK cells (11). However, by
using IFN--deficient hosts, it was found that IL-12 contributed
directly to early antimicrobial responses independent of IFN- (12, 13). Each of these properties of IL-12 may contribute to the antitumor
activity. Recently, using a complete tumor regression model, we have
analyzed the mechanism by which IL-12 induces the regression of
established murine MCA207 s.c. tumors <10 mm in size (5). Our results
indicate that IL-12 induces a Th1 cell-directed, macrophage-effected,
delayed-type hypersensitivity (DTH) response at the site of the
regressing tumors. The IL-12-mediated response is greatest when
initiated after the establishment of a host immune response to the
incipient tumor. Classic CTL are unlikely to be involved in tumor
regression, as tumor-immune animals fail to demonstrate detectable CTL
activity in vitro. Such a mechanism for IL-12-induced tumor regression
is consistent with the well-recognized role of IL-12 as an inducer of
Th1 responses in antimicrobial studies (6), but is contrary to the
widely held view that CTL-mediated direct lysis of tumor cells in vivo
is essential for tumor eradication. Realizing that a DTH response is effective against established MCA207 tumors, we went further in this study to enhance the IL-12-mediated antitumor response with the use of a well-known DTH-potentiating agent, cyclophosphamide (Cy) (14). We show that long term (>3 wk) established, large (15–20 mm), s.c. MCA207 tumors that are refractory to treatment with either IL-12 or Cy alone regress completely when a brief treatment with IL-12 is given within 5 days after a single dose of Cy. The regression of the large MCA207 tumors appears to be a self-sustained process that takes >4 wk to complete. This tumor regression model has provided a rare opportunity to study a complete antitumor immune response against a syngeneic, weakly immunogenic tumor. It also further demonstrates the great potential of cancer immunotherapy based on the biologic activities of IL-12.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine MCA207 sarcoma, a methylcholanthrene-induced
transplantable tumor in C57BL/6 mice, was obtained from the Surgery
Branch, National Cancer Institute (Dr. J. Yang, Frederick, MD). The
tumor was maintained free of Mycoplasma contamination. Tumor
cells were cultured from in vivo-harvested tumor implants in RPMI 1640
medium supplemented with 10% heat-inactivated, 2 mM glutamine, 100
µg/ml streptomycin, 100 IU/ml penicillin, and 5 x
10-5 M 2-ME. For tumor implantation, 5 x
105 tumor cells were injected in a 0.2-ml volume in saline
s.c. on the side of 10- to 16-wk-old female C57BL/6 mice or female
C57BL/6 IFN- gene knockout mice (The Jackson Laboratory, Bar Harbor,
ME). Tumor development and growth were followed by palpation and
measurement of perpendicular tumor diameters. Cure is defined as
complete tumor regression following treatment and the absence of
recurrent tumor for the entire follow-up period (4–6 mo).
In vivo treatment of tumors
MCA207 tumors were grown for 10 days (small tumor) to reach tumor sizes of 4 to 8 mm or for 3 to 4 wk to reach tumor sizes of 15 to 20 mm (large tumor) before the start of various treatments, as defined in the text and figure legends. Cy (Sigma Chemical Co., St. Louis, MO) and 5-fluorouracil (5-FU; U.S. Biochemical Corp., Cleveland, OH) were given at a single dose of 100 to 125 mg/kg (2.5 mg/mouse) in 0.5 ml of saline via i.p. injection. Cytokines were given by i.p. injection in 0.5 ml of saline according to the following doses and schedule: mIL-12 (Genetics Institute, Andover, MA), 500 ng, once every other day for three to five total injections; hIL-2 (Cetus), 50,000 IU/day for 10 days; mIL-4 (Biologic Response Modifier Program, National Cancer Institute), 1 µg/day for 10 days; and mIL-10 (Biologic Response Modifier Program), 500 ng/day for 10 days.
In vitro cytotoxicity assay
After lysis of RBC, spleen cells (5 x 106/ml) were cultured in vitro in RPMI medium with irradiated (20,000 rad) MCA207 tumor cells (1 x 105/ml) in 24-well plates for a total of 7 days. IL-2 was added to the culture at a final concentration of 40 IU/ml, 2 days after the start of restimulation. At the end of restimulation, cells were collected and washed extensively to remove dead cells. 51Cr-labeled MCA207 tumor cells were incubated with the restimulated spleen cells at the E:T ratios indicated in a 5-h chromium release assay. The percent specific lysis was calculated from triplicate samples as follows: ((experimental counts per minute - spontaneous counts per minute)/maximal counts per minute - spontaneous counts per minute)) x 100.
Immunohistochemistry
Tumors were resected and snap-frozen immediately. Cryostat
sections, 6 µm thick, were fixed in cold acetone, dried at -20°C,
and stored at -70°C before use. Before staining with Ab, the frozen
sections were dried at room temperature. After rehydration in PBS, the
sections were blocked with 1% normal goat serum and 1% BSA in PBS for
20 min. The sections were stained with primary Abs diluted in blocking
solution for 30 min at room temperature or overnight at 4°C. The
primary Abs and the working dilutions used in this study are as
follows: partially diluted rabbit anti-mouse macrophage iNOS
polyclonal serum (1/2000; Calbiochem, San Diego, CA); partially
purified rabbit anti-mouse IP-10 (1/4000; Dr. Farber, National
Institute of Allergy and Infectious Diseases, Bethesda, MD);
biotinylated rat anti-mouse CD11b/Mac-1 (5 µg/ml; clone M1/70,
PharMingen, San Diego, CA); and purified monoclonal rat anti-mouse
CD4 (15 µg/ml; clone H129.19), CD8 (15 µg/ml; clone 53-6.7),
IFN- (2.5–5 µg/ml; clone R4-6A2), IL-4 (10 µg/ml; clone 11B11),
and isotype-matched control rat IgG (10–40 µg/ml; PharMingen).
Biotinylated goat anti-rabbit IgG (1/1000; Vector Laboratories,
Burlingame, CA) and goat anti-rat IgG (5 µg/ml; PharMingen) were
used as secondary Abs for detection of unlabeled primary Abs followed
by the alkaline phosphatase-conjugated ABC system (Vector
Laboratories). Color was developed using the Vector Red substrate
(Vector Laboratories). The sections were then counterstained with
hematoxylin, cleared with xylene, and mounted permanently.
Isotype-matched control Abs/sera were also used to ensure that the
positive staining obtained with each Ab was Ag specific.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies by us (5) and others (2) have indicated that
IL-12 treatment can induce complete regression of small (<10 mm)
MCA207 tumors that are established for a short period of time (7–10
days). When tested on larger tumors (>10 mm) grown for >12 days,
treatment with IL-12 alone causes inhibition of tumor growth, but is
unable to induce complete tumor regression. Since we have previously
identified a Th1 cell-directed DTH immune response associated with the
regression of small MCA207 tumors treated with IL-12 alone, we asked
whether Cy, a well-known DTH-potentiating agent (14), can enhance the
antitumor efficacy of IL-12 in a large MCA207 tumor model. In several
experiments, summarized in Table I
, we
tested the antitumor effects of Cy and IL-12 in combination on MCA207
tumors that had been established for 24 to 34 days and were 12 to 28 mm
in diameter. The responses of tumors to various treatments listed in
Table I are categorized into four types as defined in Table I. As Table I shows, all untreated tumors grew progressively. Treating large tumors
with Cy alone caused a transient tumor regression that lasted for 2 wk,
and all tumors resumed growth subsequently. On the other hand,
treatment of large tumors with IL-12 alone only minimally inhibited
tumor growth for a short period (3–7 days). The few tumors that
regressed with IL-12 treatment were smaller ones with sizes near 12 mm
at the beginning of the treatment. Among mice treated with the
combination of IL-12 and Cy, when IL-12 was administered 1, 4, or 5
days, but not 7 or 10 days, after a single dose of Cy, the majority of
tumors were completely eradicated. The few tumors that did not regress
completely were the largest ones with sizes >22 mm in diameter at the
initiation of treatment. In contrast, the curative effect of Cy and
IL-12 in combination was lost if IL-12 was given before, instead of
after, the single dose of Cy (Table I, IL-12, 10 days, Cy). Following
the initial three to five doses of IL-12 given over 5 to 7 days, a
single dose of IL-12 was again administered to some tumor-bearing
animals 7 to 14 days after the previous IL-12 injection. This
"maintenance" injection was especially necessary for complete
eradication of larger (>18 mm) tumors. Regression of these tumors
slowed down and stopped if the maintenance IL-12 injection was withheld
(not shown). In most instances, complete tumor regression took
approximately 1 mo or longer to complete. The patterns of tumor
responses to some of the treatments summarized in Table I are
illustrated in Figure 1. Untreated tumors
grew rapidly to reach sizes of >500 mm2. Treatment of
similar sized tumors (>15 mm in diameter) with IL-12 alone barely
caused an inhibition of tumor progression during the first week of
IL-12 administration, and progressive tumor growth followed. Cy alone,
as summarized in Table I, induced transient tumor regression that
lasted for 2 wk, and then all tumors resumed growth. Finally, the
combination treatment of a single dose of Cy followed by IL-12 induced
continued tumor regression until complete eradication occurred 1 mo
later. Mice that completely eradicated tumor were resistant to
subsequent rechallenge with MCA207 tumor cells, indicating the presence
of an immunologic memory.
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indicates, tumors treated with a single dose of 5-FU followed by IL-12
did not show any regression, indicating that the contribution of Cy to
the success of the combination treatment with IL-12 is most likely due
to its immunopotentiating activity rather than to any direct tumor
cytotoxicity. The contribution of IL-12 to the success of tumor
eradication is also unique. As Table I shows, tumor eradication was not
seen with any of the other three cytokines tested, IL-2, IL-4, or
IL-10. The antitumor activity of Cy alone in the MCA207 tumor model
Since Cy alone was able to induce transient regression of large
MCA207 tumors (Table I and Fig. 1), and it has been reported to induce
complete tumor regression in other studies (16, 17), we tested the
antitumor activity of Cy in the 3- and 10-day established small MCA207
tumor models. Similar to a previous report by others in a different
tumor model (16), treatment of 3-day established nonpalpable MCA207
tumors resulted in only a delay in tumor appearance, while treating
palpable tumors (4–8 mm) 10 days after tumor implantation resulted in
initial regression of all tumors and complete eradication of tumor in
12 of 17 mice (Table II). Since mice with
greater (10-day established) tumor burdens responded better to Cy
treatment than those with lesser (3-day nonpalpable) burdens, it
suggests that direct cytotoxicity of Cy was not the main effector
mechanism for tumor regression as was also suggested in previous
studies (16, 17). Finally, similar to observations in another tumor
model treated with Cy (16), MCA207 tumors initially treated with Cy 3
days after tumor inoculation did not respond to a subsequent second
treatment when the previously treated tumors became palpable
(Table II).
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dependence of Cy- and IL-12-mediated tumor regression
Previous studies have shown that regression of MCA207 tumors
induced by IL-12 requires the presence of IFN- (2). We confirmed
this finding with the use of IFN- gene knockout mice in the 10-day
MCA207 tumor regression model. In this experiment three of five normal
mice bearing 6- to 10-mm tumors treated with IL-12 alone were rendered
tumor free, while none of nine tumors (6–12 mm) in the IFN- gene
knockout mice had a complete response (Table III). Although previous studies (18) and
our preliminary results (data not shown) have demonstrated that daily
injection of high doses of IL-12 (500–1000 ng/day) caused significant
treatment mortality within 1 wk in IFN- gene knockout mice, the lack
of response to IL-12 treatment in IFN- gene knockout mice in this
experiment was not due to treatment-associated toxicity, because no
toxicity was observed when 500 ng/injection of IL-12 was administered
i.p. every other day for three doses. In contrast, Cy treatment of
IFN- knockout mice bearing 10-day established MCA207 tumors resulted
in the same pattern of regression as that seen in normal mice and tumor
eradication, albeit at a lower rate (20%) than that in normal mice
(70%; Table III), was achieved in some animals. Further, IFN- gene
knockout mice that were rendered tumor free by Cy treatment were
resistant to subsequent challenge with 1 x 106
MCA207 tumor cells 1 mo later, indicating the presence of T
cell-mediated immune memory independent of IFN-. We next studied the
combination of Cy and IL-12 in the IFN- gene knockout mice with
large MCA207 tumors. As indicated in Table III, the initial transient
regression of MCA207 tumors did take place after Cy treatment, but the
tumor resumed growth after 12 days despite the combined treatment with
IL-12, suggesting that tumor eradication by the Cy and IL-12
combination treatment is dependent on IFN-.
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We (5) and others (19) have reported extensive iNOS expression by
activated macrophages at the site of tumors treated with IL-12 alone.
Other recent studies have suggested the presence of another putative
effector molecule, IP-10, associated with tumor regression induced by
IL-12 (20). In this study we examined the expression of both iNOS and
IP-10 by macrophages in MCA207 tumors treated with Cy alone, IL-12
alone, and Cy plus IL-12. Compared with the peripheral presence of
macrophages in the untreated tumors (Fig. 2, day 0, Mac-1), in the small regressing
MCA207 tumors treated with Cy alone, massive macrophage infiltration,
known to be the hallmark of Cy treatment of tumors (21, 22), appeared
as early as 2 days after Cy treatment (Fig. 2, day 2, Mac-1) and
remained in the regressing tumor throughout the entire period (9 days)
of tumor regression. Despite this massive macrophage infiltrate, no
expression of iNOS was detected throughout the entire period of tumor
regression (Fig. 2, iNOS, days 2–9), and mild, but clear, expression
of IP-10 by infiltrating macrophages was seen at all stages of tumor
regression (Fig. 2, IP-10, days 2–9). To determine whether IP-10 is
also involved in IL-12-mediated small tumor regression, IP-10 staining
was repeated on samples collected from the previous study in which
extensive iNOS expression by macrophages in the small regressing MCA207
tumors treated with IL-12 was observed (5). Despite the presence of
massive numbers of activated macrophages and clear expression of iNOS
(5), only sporadic expression of IP-10 by macrophages was seen (not
shown), suggesting that IP-10 is unlikely to be essential for the
regression of small MCA207 tumors treated with IL-12 alone. Thus,
evidence collected to date indicates that nitric oxide is unlikely to
be an effector molecule in Cy-mediated tumor regression, and IP-10 may
be involved in the regression of tumors treated with Cy but not
IL-12.
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A) and in
the large tumors treated with Cy for 4 days before initiation of IL-12
(Fig. 3B) or in tumors treated with Cy alone for 8
days (Fig. 3C). The iNOS expression was not obvious 2
days after the start of IL-12 treatment (Fig. 3D),
but became consistently detectable in the tumors at all stages of tumor
regression 4 days after the start of IL-12 treatment (Fig. 3, E-G, I, and K). Note that the
tumors shown in Figure 3, C and E, were both
treated with Cy for 8 days, except that the tumor shown in Figure 3E had received additional IL-12 treatment 4 days after Cy,
indicating that IL-12 administration was responsible for the observed
iNOS expression. Similar to the previous observation, iNOS was mainly
expressed by macrophages (Mac-1; compare Fig. 3, I and
J). Interestingly, in the large MCA207 tumor model,
the expression of iNOS seemed to correlate with the use of IL-12 rather
than with the regression of the tumors, as a nonregressing tumor
treated with IL-12 alone 15 days after the start of the treatment also
showed intense local expression of iNOS (Fig. 3H).
Even 25 days after the start of IL-12 treatment, some expression of
iNOS, albeit limited in locality, was still visible in progressively
growing tumors treated with IL-12 alone (Fig. 3L). In
contrast to the lack of iNOS expression in tumors treated with Cy alone
(Fig. 3, B and C), extensive IP-10
expression by infiltrating lymphocytes was seen associated with the
heavily necrotic area of large tumors treated with Cy 4 and 8 days
previously (Fig. 4, B and
C). The IP-10-expressing lymphocytes found in the
heavily necrotic area of the Cy-treated tumors were probably
macrophages, as they stained positive for the Mac-1 marker (match Fig. 4, K and L). Interestingly, this tumor
necrosis with disrupted tumor cell nuclei and a heavy cellular
infiltration of macrophages expressing IP-10, but not iNOS, was only
seen in Cy-treated large tumors. In the same size tumors treated with
Cy plus IL-12, there was an increase in iNOS (Fig. 3, E vs
C) and a down-regulation of IP-10 (Fig. 4, E vs C) expression. This down-regulation
of IP-10 expression was obvious as early as 2 days after the start
of IL-12 treatment at which time no iNOS expression could be detected
(Figs. 3D and 4D). In contrast to the
association of iNOS expression with IL-12 treatment of tumors (Fig. 3H), no clear IP-10 expression was seen in tumors
treated with IL-12 alone (Fig. 4H). This is
consistent with findings from small regressing tumors treated with
Cy or IL-12 (Fig. 2).
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). Surprisingly,
splenocytes from Cy-cured IFN--deficient animals that had resisted
the subsequent challenge with MCA207 tumors also failed to demonstrate
CTL activity (Fig. 5). The lack of demonstrable CTL activity in these
two cases was not due to a failure of experimental procedures, as
splenocytes from animals rendered tumor free by Cy plus IL-12 treatment
assayed in the same experiment did show appreciable levels of CTL
activity at a E:T ratio of 40:1 (27%; Fig. 5). Thus, it appears that a
measurable CTL response was activated during the regression of large
MCA207 tumors treated by Cy plus IL-12, but not in small regressing
MCA207 tumors treated with either Cy or IL-12 alone. Further, despite
the fact that the IFN- gene knockout mice were able to reject a
subsequent tumor rechallenge following Cy-induced complete eradication
of small tumors, we did not detect any CTL activity in these mice (Fig. 5). Therefore, it remains to be determined how an immunologic memory
against MCA207 tumor independent of both CTL and IFN- is established
and functional in the IFN- gene knockout mice treated with Cy
alone.
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To determine whether tumor regression induced by Cy plus IL-12
also involves a Th1 cell-directed immune response as was seen in the
regression of small MCA207 tumors treated by IL-12 alone (5), tumor
samples collected before and at various time points after the start of
Cy plus IL-12 treatment were stained with Abs to CD4, CD8, IFN-, and
IL-4 (Fig. 6). Untreated MCA207 tumors
grown in vivo for >3 wk contained few CD4 T cells, which were
identified mainly at the periphery, and some single CD8 T cells located
both at the periphery and within the tumor; these were only seen under
high magnification (x400; Fig. 6). While a few T cells at the
periphery expressed either IL-4 or IFN- (not shown), none of the T
cells within the tumor expressed either cytokine (Fig. 6, day 0,
IFN- and IL-4). Treatment of the large tumors with a single dose of
Cy resulted in the total loss of T cells (both CD4 and CD8 cells) in
the tumor both 4 (not shown) and 8 days after Cy treatment (Fig. 6, Cy,
day 8, CD4 and CD8). Despite this total lack of T cells in the
Cy-treated tumor, expression of both IFN- and IL-4 by many
infiltrating mononuclear lymphocytes was found. While IFN- was
expressed in both the necrotic (Fig. 6, Cy, day 8, IFN-) and the
nonnecrotic (not shown) areas of the tumor, IL-4 was mainly expressed
by lymphocytes at the boundary between necrotic and nonnecrotic areas
(Fig. 6, Cy, day 8, IL-4). Staining with Ab to the macrophage marker
Mac-1 indicated that many of the cytokine-expressing lymphocytes were
macrophages (not shown). It should be pointed out that the same
Mac-1+ cells in the necrotic areas of Cy-treated
tumors that expressed IFN- (Fig. 6, Cy, day 8, IFN-) also
expressed IP-10 (Fig. 4, K and L). Next,
with the administration of IL-12 4 days after Cy, T cell recovery in
the treated tumors was rapid. Thus, 2 days after the start of IL-12
following Cy treatment, clusters of CD4 T cells appeared at the
periphery, and a few single CD8 T cells appeared within the tumor (Fig. 6, Cy plus IL-12, days 6/2, CD4 and CD8). As in the tumors treated only
with Cy, both IFN- and IL-4 were expressed by many
Mac-1+ cells in the nonnecrotic areas, and the expression
of these cytokines by the newly recovered T cells was not obvious (Fig. 6, Cy plus IL-12, days 6/2, IFN- and IL-4). By 4 days after the
start of IL-12 treatment (or 8 days after Cy), massive CD4 and CD8 T
cell infiltrates were found under lower (x100) magnification (Fig. 6, Cy plus IL-12, days 8/4, CD4 and CD8), forming a clear contrast to the
lack of T cells in tumors treated with Cy alone 8 days earlier (Fig. 6, Cy, day 8, CD4 and CD8). Besides the macrophages, many of the
infiltrating T cells also expressed IFN-, while IL-4 was mainly
expressed by non-T cells (Fig. 6, Cy plus IL-12, days 8/4, IFN- and
IL-4). The expression of both IFN- and IL-4 by non-T cells decreased
significantly 4 days later; thus, by 12 days after Cy treatment or 8
days after the start of IL-12 treatment, T cell-mediated cytokine
expression with an imbalance toward more production of IFN- became
obvious in the tumors (not shown). A clear dominance of IFN- and
lack of IL-4 production by T cells, the hallmark of a Th1 response,
took place during the third week following the start of treatment (Fig. 6, Cy plus IL-12, days 19/15, IFN- and IL-4). Also at this time,
peak numbers of CD4 and CD8 T cells were seen in the regressing tumors
(Fig. 6, Cy plus IL-12, days 19/15, CD4 and CD8; note that a low
magnification of x40 was used). This time period coincided with the
regrowth of tumors treated with Cy alone following a transient
regression and a continued rapid regression of tumors treated with Cy
plus IL-12 (Fig. 1). On the other hand, tumors treated with IL-12 alone
without Cy failed to show any regression over the pretreatment sizes
(Fig. 1), and these tumors contained T cell infiltrates limited in
location and number (Fig. 6, IL-12, day 15, CD4 and CD8; note that a
higher magnification of x100 was used). These tumors also lacked
intratumoral cytokine production (Fig. 6, IL-12, day 15, IFN- and
IL-4). For the Cy- plus IL-12-treated tumors, the last week of tumor
regression was associated with a gradual diminution of T cell
infiltrates (Fig. 6, Cy plud IL-12, days 29/25, CD4 and CD8). However,
not proportional to the reduction of T cells was the almost complete
cessation of IFN- production and the appearance of IL-4 production
by a few T cells (Fig. 6, Cy plud IL-12, dayd 29/25, IFN- and
IL-4).
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and IL-4 were produced, mainly by non-T cells.
During the midphase and at the peak of tumor regression, a Th1 cytokine
response dominated. In the final phase toward the completion of tumor
eradication, IFN- production was turned off, and IL-4 production by
some T cells occurred. Expression of other cytokines in the regressing
tumors were also analyzed. Similar to the observation in the small
MCA207 tumor model (5), we did not see any significant expression or
increase in levels of expression of IL-2 and IL-10 at any time point
analyzed (not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The essential role of IL-12 in this large tumor regression model
is supported by the fact that other cytokines (IL-2, IL-4, and IL-10)
combined with Cy did not have similar antitumor effects (Table I). We
chose IL-4 and IL-10 because these cytokines are instrumental in Th2-
and CTL-mediated immune responses and IL-2 because Cy had been shown
previously to augment the IL-2-mediated antitumor effect. With the same
dose of IL-4 used in our experiment, others have shown a significant
antitumor effect by systemic administration (28). Although
enhanced antitumor effects have been previously reported with the
combination of Cy and IL-2 (29, 30), the tumors were small (<8 mm). In
the current study the combination of Cy and IL-2 was unsuccessful in
eradicating large tumors (Table I). The unique requirement for IL-12 is
consistent with the mechanism of a Th1 cell-directed immunity
responsible for tumor rejection as further identified in this study.
This Th1 response, earmarked by the T cell-mediated tumor site
production of IFN-, but not IL-4, was first observed during the
return of massive T cell infiltrates to the treated tumors 4 days after
the start of IL-12 and peaked in the third week after the start of the
Cy plus IL-12 treatment (Fig. 6). Unlike the Th1 response first
observed in the small regressing MCA207 tumors treated with IL-12 alone
(5), predominant production of IFN- by T cells did not continue
until the complete eradication of large tumors treated with Cy plus
IL-12. During the late phase of tumor regression, IFN- production
decreased to undetectable levels despite the persistent presence of
large numbers of T cell infiltrates (Fig. 6). It is not clear whether
another type of T cell response substituted for the early Th1 response
and was responsible for final eradication of the tumor. Since CTL
activity was detectable from animals rendered tumor free by treatment
with Cy plus IL-12 in this study (Fig. 5), one possibility is the
development of a CTL response for complete tumor eradication during the
late phase of tumor regression. Future experiments will address this
issue.
Since a similar Th1-mediated antitumor response has been identified in
both the small (5) and the large (this study) MCA207 tumor regression
models, it appears that rather than activating a different antitumor
immune response, the role of Cy is to facilitate the onset of an
IL-12-dependent Th1 response. As shown in this study, in the absence of
Cy, IL-12 alone was unable to induce the regression of large MCA207
tumors (Table I and Fig. 1). Comparison of regressing tumors treated
with Cy plus IL-12 and progressing tumors treated with IL-12 alone
revealed a lack of sufficient T cell infiltration as well as tumor site
IFN- production by the infiltrating T cells in animals treated with
IL-12 alone (Fig. 6, Cy plus IL-12, days 19/15 vs IL-12, day 15). The
presence of large tumors may suppress the induction of a Th1 response
that can be activated efficiently in animals bearing small tumors by
IL-12 alone. Pretreatment of the same large tumors with Cy appears to
help the initiation of this response. How does Cy treatment facilitate
the initiation of the Th1 response? Several possible actions of Cy may
account for this effect. First, Cy treatment may reduce the tumor
burden significantly so that IL-12 alone is able to initiate a potent
Th1 response to a smaller tumor. This is unlikely in light of the fact
that IL-12 treatment must be initiated within 5 days after Cy
treatment, at which time tumor regression has just begun, but not at
the time when the tumor is the smallest (8–14 days, Fig. 1). Second,
Cy treatment may remove the so-called suppressor T cells. This
mechanism of Cy remains possible but uncertain, because there has not
been any clear identification or characterization of suppressor T cells
in any tumor model. Unlike the untreated small MCA207 tumors that
contain Th2 cells that preferentially express IL-4 (5), untreated large
MCA207 tumors do not contain large numbers of T cells, and among the T
cells associated with the tumor, no clear pattern of a Th1 or a Th2
response has been observed (Fig. 6). Thus, it is also unlikely that Cy
facilitates the initiation of a Th1 response by eliminating a
predominant Th2 response before IL-12 treatment. Third, Cy treatment
may affect the presentation of tumor Ags that, in turn, affects T cell
activation. This is highly possible because the direct cytotoxicity of
Cy can generate large number of dead tumor cells, and tumor debris can
be picked up by infiltrating macrophages promoted by Cy treatment.
Finally, cytokine production by macrophages associated with Cy
treatment as revealed in this study may also affect the course of a
subsequent T cell response. Both IFN- and IL-4 were produced by
macrophages infiltrating the Cy-treated large tumors (Fig. 6, Cy, day
8). Since only IFN-, not IL-4, production was observed in the
necrotic area of the Cy-treated tumor, it seems that the cytokines were
not simultaneously produced by a single macrophage. As a result, the
preferential production of IFN-, but not IL-4, in the necrotic area
of the tumors may in some way favor the induction of a Th1, but not a
Th2, response. In this respect it would be interesting in future
experiments to see whether induction of a Th1 response by Cy plus IL-12
is interrupted in mice lacking the IFN- gene.
As in our previous study (5), the presence of a pre-existing host T
cell response to the incipient tumors or, in other words, the presence
of a tumor-specific memory seems to play an important role in an
effective T cell response activated by Cy plus IL-12. Since it has been
shown that Cy treatment can eliminate primary, but not secondary, T
cell responses (31, 32), and since direct cytotoxicity of Cy to T cells
(33, 34) was confirmed by the total lack of T cells in Cy-treated
tumors for at least 8 days (Fig. 6, Cy, day 8), it is likely that the
tumor-infiltrating T cells found after treatment with Cy and IL-12 were
derived from memory T cells that had survived the cytotoxicity of Cy
(32). Such a pre-existing host response, albeit not able to reject the
incipient tumor, is essential for endotoxin-induced tumor regression,
and it has also been implicated as the major factor distinguishing a
responder from a nonresponder (26). In this study the known
cytotoxicity of Cy toward the majority of T cells makes priming of
tumor-specific T cells from naive precursors following Cy treatment
unlikely. Instead, the rapid recovery of T cells in tumors treated with
both Cy and IL-12 (Fig. 6, Cy plus IL-12, days 6/2–8/4), but not in
tumors treated with Cy alone (Fig. 6, Cy, day 8), further argues for
the involvement of memory, but not naive, T cells as targets of IL-12
action in the MCA207 tumor regression model. Consistent with this view,
one recent study indicates that IL-12 promotes the proliferation of
Ag-stimulated CTL, but not unstimulated T cells (35). If, indeed, the
pre-existing T cell response is a major factor influencing the
likelihood of a tumor’s response to IL-12-based treatment, the
question then is how to detect this pre-existing immunity before
treatment. In this regard, the appearance of cytokine-producing T cells
associated with the tumor before the start of treatment may be an
indicator of such a pre-existing host T cell response. Future
experiments will focus on the comparison of pretreatment cellular and
cytokine profiles of IL-12-responding and nonresponding tumors to
determine whether a marker for the likelihood of a treatment response
can be identified.
In conclusion, we have described a large tumor treatment model in which MCA207 tumors grown >3 wk to sizes as large as 20 mm in diameter can be completely eradicated by treatment with Cy and IL-12. This model offers an opportunity to study the immune response capable of eradicating large, weakly immunogenic tumors. In addition, the model extends previous findings concerning the antitumor activity of IL-12 by demonstrating that IL-12 activity can be dramatically enhanced by DTH-potentiating agents such as cyclophosphamide. Through better understanding of the IL-12-mediated antitumor immune response as seen in this model, it may be possible to identify factors that are either missing or inadequate in other less responsive or nonresponsive tumors. With this knowledge, it is hoped that improved antitumor immune responses may be extended to other animal tumors and, eventually, to patients with cancer.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: IP-10, INF--inducible protein 10; DTH, delayed-type hypersensitivity; Cy, cyclophosphamide; 5-FU, 5-fluorouracil; m, murine; h, human; iNOS, inducible nitric oxide synthase.
Received for publication June 6, 1997. Accepted for publication October 8, 1997.
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