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Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, and
Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and
Department of Medicine, Vanderbilt Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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knockout mice were more susceptible. Purified
CD8 T cells from CD4-depleted mice following tumor regression had more
IFN- mRNA and lysed tumor cells without stimulation ex vivo, in
contrast to CD4-intact mice. Thus, the quality as well as quantity of
CD8+ CTL determines the completeness of immunosurveillance
and is controlled by CD4 T cells but not solely Th2 cytokines. This
model of immunosurveillance may indicate ways to enhance the efficacy
of surveillance and improve immunotherapy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite the induction of a specific CTL response and appropriate help by helper T cells, it has been difficult to eradicate all of the tumor cells. There is accumulating evidence for escape mechanisms of tumor cells (17, 18, 19), including loss of Ag or class I expression, production of suppressive cytokines by tumor cells, and expression of Fas ligand on tumor cells (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). Considering the fact that escape variants expanded again after nearly complete rejection (20), it is important to determine how to prevent these tumor escape mechanisms to obtain durable remissions.
In this study, we examine whether CTL induced against a model viral tumor Ag can control the growth of tumor, how tumor cells escape from this immunosurveillance, and how we can prevent those escape variants. As a model tumor with a well-characterized Ag as a model viral tumor Ag, we used a BALB/c 3T3 fibroblast line transfected with HIV gp160 and with mutant ras and myc for tumorigenicity. Immunosurveillance of tumors expressing viral Ags may succeed, as is often the case for EBV-transformed B lymphocytes, or sometimes fail, as in the case of cervical carcinoma expressing human papillomavirus Ags, even in individuals who are not immunodeficient. We have previously characterized in depth the murine CTL response to an immunodominant determinant of gp160, called P18, contained within the V3 loop (38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48), that facilitates use of this Ag as a model viral tumor Ag.
There have been reports indicating that Th2-type cytokines down-regulated antitumor immunity (49, 50) and the activation of type 1 T cell responses produced antitumor immunity (51, 52, 53, 54). In a cross-sectional epidemiological study of papillomavirus-related cervical neoplasia, we observed an inverse correlation between the fraction of individuals making a Th1 cytokine response and the degree of progression of disease (55). Thus, a shift to Th1-type cytokine production may be one goal for effective immunotherapy for tumors as well as virus infection. Therefore, we also address a question whether reduction of Th2-type cytokine production could enhance immunosurveillance. We found a novel striking enhancement of surveillance by CD4 cell depletion that cannot be explained primarily by elimination of Th2 cells.
Thus, we have taken advantage of the intriguing pattern of growth,
spontaneous (immune-mediated) regression, and recurrence of this novel
model tumor to investigate the relation between different cellular
immune responses and tumor growth. We examined the role of CD8 and CD4
cells in the initial tumor regression and in preventing or facilitating
tumor recurrence. We also examined the molecular mechanism of tumor
escape from CTL in vivo. Unexpectedly, we discovered a novel important
role for the quality of CTL, with respect to ex vivo activity and the
amount of IFN- mRNA, in prevention of recurrence of tumor, and the
regulation of this CTL quality by CD4 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice were purchased from Charles River Breeding
Laboratories (Frederick, MD). IL-4 knockout
(KO)2 and IFN- KO
mice having the BALB/c background were obtained from The Jackson
Laboratory (Bar Harbor, ME). All mice were kept under pathogen-free
conditions and used at 6–10 wk old. Animal experiments were all
approved by the National Cancer Institute (NCI) Animal Care and Use
Committee.
Anti-CD4 mAb (clone GK1.5) and anti-CD8 mAb (clone 2.43) for in
vivo depletion were obtained from the Frederick Cancer Research and
Development Center, NCI (Frederick, MD). FITC-conjugated
anti-H-2Dd mAb (clone 34-2-12) was purchased
from PharMingen (San Diego, CA). Mouse CD8 T cell subset enrichment
columns were obtained from R&D Systems (Minneapolis, MN). Recombinant
vaccinia vPE16, expressing the gp160 envelope protein of HIV-1 strain
IIIB and control vaccinia vSC8, expressing ß-galactosidase, were
kindly contributed by Drs. Patricia Earl and Bernard Moss (National
Institute of Allergy and Infectious Disease, Bethesda, MD) (56, 57).
Trizol Reagent, Super Script cDNA synthesis reagents, and PCR
SuperMixture were purchased from Life Technologies (Rockville, MD).
NorthernMax, Stripe-EZ RNA Kit, and pT7 mouse IFN- probe template
were obtained from Ambion (Austin, TX).
Tumor cells
15-12 are BALB/c 3T3 cells transfected with gp160 envelope protein of HIV-1 IIIB (38). 18Neo are BALB/c 3T3 cells transfected with the neomycin resistance gene alone as a control. 15-12RM was made from 15-12 by transfection with Myc and mutant Ras genes, containing the substitution of glycine to valine at position 12 of K-ras p21. All cells were maintained in T cell complete medium containing 0.2 mg/ml of geneticin (Sigma, St. Louis, MO).
CTL generation
Splenocytes from BALB/c mice previously immunized with 1 x 107 PFU of vPE16 were stimulated with irradiated BALB/c splenocytes pulsed with 1 µM peptide18 (P18) IIIB in a 24-well culture plate in complete T cell medium supplemented with 10% T-stim (Collaborative Biomedical Products, Bedford, MA). After 7 days of culture, viable cells were harvested and a CTL line against P18-IIIB was established by several restimulations with P18-IIIB-pulsed splenocytes. A CTL line for 15-12RM was derived from splenocytes of 15-12RM tumor-bearing mice taken on day 50 after inoculation of 15-12RM cells. These splenocytes were stimulated with 15-12RM cells, which were treated with mitomycin C (Sigma) (100 µg/ml for 45 min), plus irradiated splenocytes of normal BALB/c mice. The CTL line specific for 15-12RM was induced after several such restimulations. T cell complete medium is Biofluids (Rockville, MD) R2E Medium (a 50:50 mixture of RPMI 1640 and EHAA media) supplemented with L-glutamine, sodium pyruvate, nonessential amino acids, penicillin, streptomycin, 5 x 10-5 M 2-ME, and 10% FCS.
CTL assay
Cytolytic activity against several target cells was assayed by a 4-h 51Cr-release assay, as described elsewhere (58). Tumor cells harvested from tumor-bearing mice were used for target cells either on the same day when they were resected or after 1 wk of culture in complete T cell medium containing geneticin. Where indicated, CD8+ T cells were purified from splenocytes using a mouse CD8+ T cell subset enrichment column (R&D Systems) and used as effector cells. The percentage of specific 51Cr release was calculated as: 100 x (experimental release - spontaneous release)/(maximum release - spontaneous release). Maximum release was determined from supernatants of cells that were lysed by addition of 5% Triton X-100. Spontaneous release was determined from target cells incubated without added effector cells.
Tumor inoculation
A total of 1 x 106 15-12RM cells in 200 µl of PBS were injected s.c. on the right flank of the mouse. Where indicated, 1 x 107 PFU of vPE16 or vSC8 were injected i.v. at 5 wk and 7 days before tumor inoculation. Some mice were treated i.p. with 0.2 ml of PBS containing either 0.5 mg of anti-CD4 mAb (clone GK1.5), anti-CD8 mAb (clone 2.43), or control rat IgG (ICN Pharmaceuticals, Costa Mesa, CA) starting 3 days before tumor cell injection and then twice a week.
Detection of mRNA by RT-PCR and Northern blot analysis
Total RNA was extracted from 5 x 106
tumor cells in Trizol reagent (Life Technologies). cDNAs were
synthesized by extension of oligo(dT) primers using the Super Script
Preamplification System (Life Technologies), according to the
manufacturer’s instructions. PCR of the cDNA was performed in a final
volume of 50 µl containing each primer at 0.2 µM and Supermixture
(Life Technologies) using the GeneAmp 9700 PCR system (Perkin-Elmer,
Norwalk, CT). The amplification cycles were 94°C for 30 s,
55°C for 1 min, and 72°C for 1 min. After 25 cycles, PCR products
were separated by 10% TBE gel electrophoresis and stained with Vista
Green (Amersham, Arlington Heights, IL). The sequences of primers are
as follows: hypoxanthine-guanine phosphoribosyl transferase (HPRT)
(sense), 5'-GTTGGATACAGGCCAGACTTTGTTG-3'; HPRT (antisense),
3'-GAGGGTAGGCTGGCCTATGGCT-5'; gp160 v3 loop (sense),
5'-GCTGTTAAATGGCAGTCTAGC-3'; gp160 v3 loop (antisense),
3'-CGTTAGGAGTCCTCCCCTGGG-5'. Northern blot analysis was performed
using NorthernMax and Strip-EZ RNA Kit (Ambion), according to the
manufacturer’s instruction. Total cellular RNA was used, and the
integrity of RNA was tested by electrophoresis in 2% agarose gel. A
total of 15 µg of RNA was loaded per lane and transferred onto nylon
membranes. After prehybridization, the
32P-labeled probe for IFN- was hybridized at
65°C overnight. Then the membrane was washed with low- and
high-stringency solution. Autoradiography was conducted at -70°C for
up to 4 days by using Kodak (Rochester, NY) BioMax-MS film. The same
membrane was stripped and used to rehybridize with a GAPDH probe as an
internal control.
Flow cytometry
18Neo and tumor cells were stained with FITC-conjugated anti-H-2Dd mAb to detect the expression of class I molecules. They were analyzed by FACScan using CellQuest software (both from Becton Dickinson, Mountain View, CA).
Histological examination
Tumor tissues were excised on the indicated days after inoculation of tumor cells, fixed in 10% v/v formalin solution, and processed for paraffin embedding. Sections were cut according to standard procedures and stained with hematoxylin and eosin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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15-12RM cells were injected s.c. on the right flank at day 0.
Tumors initially started growing within 5 days after inoculation, and
they reached about 8–10 mm diameter at approximately day 7. The tumors
then began to regress spontaneously and disappeared after 
10–12
days. The growth rate decreased at this time, even in the occasional
small tumors that remained. However, the mice in which tumors had
regressed initially, even beyond the point of detection, developed
tumors again between 20 and 30 days after inoculation. These did not
regress in this second growing stage (Fig. 1
, a and b).
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c). These vPE16-immune mice also had
CD8+ CTL specific for HIV-1 gp160 peptide
P18-IIIB (data not shown, and Refs. 38 and 48). Thus, immunization
against gp160, inducing specific CTL, could protect mice from the
initial growth of tumors, but not from the later development observed
30 days after inoculation of tumor cells. Regression depends on the existence of CD8 T cells
From the above results, we hypothesized that
CD8+ CTL might play a role in the initial
regression of tumor. To investigate the role of CD4 and CD8 T cells in
the regression and growth of tumor, nonimmunized mice were treated with
Abs against CD4 or CD8 molecules. Flow cytometric analysis showed that
>98% of CD4 and CD8 T cells were depleted by the treatment with the
respective Abs (data not shown). In the anti-CD8 Ab-treated groups,
tumors grew initially in the same time period as in control mice, but
they continued growing without regression (Fig. 2a). Their growth rate did not
decrease at 10–12 days, when the tumors of control mice regressed.
Although anti-CD4 Ab treatment did not result in any significant
change in the process of growth and regression of tumors up to day 30
after the challenge with tumor cells, depletion of CD4 cells
unexpectedly protected the mice from later regrowth of tumor (Fig. 2a). When CD8 cells were depleted simultaneously with
depletion of CD4 cells, the tumors continued to grow without
regression, as in the anti-CD8 Ab-injected group (Fig. 2b). Thus, CD8 cells were necessary for initial rejection of
the tumor, and the depletion of CD4 cells somehow prevented the
regrowth of tumors after regression. When anti-CD8 Ab were given to
the CD4-depleted group only after regression (starting from day 21),
tumors developed in only some of the mice, despite the fact that there
were no CD8 cells (data not shown). This observation is consistent with
the interpretation that, in anti-CD4 treated mice, 15-12RM tumor
cells did not survive after the initial regression, so the absence of
CD8 cells at the later time point was no longer relevant.
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Since it was suggested that the growth and regression of tumors
were regulated by immunosurveillance, especially CD8 T cells, we
performed a histological examination of tumors to see whether they were
compatible with the observation described above. Specimens were
harvested from 15-12 RM tumor cells injected into normal mice at
different time points after inoculation. At day 9, when initial tumors
were growing, focal sparse mixed cellular (chronic) inflammatory
infiltrates were noted within the tumor mass. At day 13, when growth
stopped, the number of infiltrating lymphocytes seen around tumor cells
increased (Fig. 3), and, when there were
no tumors on the surface of the flank at day 19, the lymphocytic
response reached a maximal level (Fig. 3). These findings raise the
possibility that these infiltrated lymphocytes contributed to the
regression of tumors. In contrast, when tumor reappeared and grew again
after regression, the recurrent tumors had a more spindle-shaped
morphology and were relatively devoid of infiltrating lymphocytes at
days 33 and 46 (Fig. 3). Thus, the histology was consistent with a lack
of a cellular immune response to the recurrent tumors.
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CTL specific for 15-12 RM tumor could also lyse target cells
presenting P18-IIIB from the v3 loop of the HIV-1 envelope (Fig. 4b). Likewise,
P18-IIIB-specific CTL, which were induced from a vPE16-immunized mouse
by stimulation with 1 µM P18-IIIB-pulsed spleen cells, could
recognize 15-12 RM tumor cells (Fig. 4a). Therefore, we
utilized CTL against both 15-12 RM tumor cells and P18-IIIB to
determine the susceptibility of the tumor cells to cytotoxicity. Our
objective was to examine how tumors recurred after initial regression,
despite the rejection mediated by CD8 T cells or the immune protection
against initial growth observed in vPE16-immunized mice. The possible
explanations of this observation could be either alteration of tumor
cells (escape variants) or induction of tolerance in responding T
cells. To examine the former possibility, tumor cells were recovered
from mice at different stages in vivo and used as target cells in the
CTL assay. While the tumor cells that grew initially retained
sensitivity for 15-12 RM CTL, tumor cells from the recurrent stage
could not be lysed by the same CTL (Fig. 5, a and b). Both
freshly isolated cells and cultured cells, which were selected by
growth in culture medium containing geneticin (to kill cells that had
lost the NeoR gene), showed the same
results. As mentioned above, vPE16-immunized mice developed tumors late
without an early growth and regression phase. Tumor cells from these
animals were not killed by 15-12 RM CTL (Fig. 5a). However,
tumor cells from normal or vSC8-immunized mice treated with
anti-CD8 mAb were killed by 15-12 RM CTL (Fig. 5c).
Moreover, 15-12RM-specific CTL could kill tumor cells harvested from
vPE16 immunized, anti-CD4 plus anti-CD8 mAb-treated mice (data
not shown).
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, 15-12RM were lysed by P18 CTL, even
after two rounds of selection, in three of four experiments. Moreover,
in the one case when cells became resistant after killing by CTL,
although they were lysed after first round killing at the same level as
original 15-12RM, they showed <5% of lysis after the second round.
Therefore, the data suggest that it is unlikely that preexisting
resistant tumor cells grew after regression of susceptible tumor cells
in vivo. In summary, 15-12 RM cells before the tumor regression caused
by CD8 T cells were susceptible to lysis by 15-12 RM CTL, but they did
not maintain this character when they recurred after initial
regression. Although vPE16-immunized mice showed early protection
against tumor growth instead of growth and regression of tumor, 15-12
RM tumors that grew out in these mice after day 30 were resistant to
gp160-specific CTL.
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To explore the mechanism of tumor resistance to CTL lysis, we
examined the expression of the class I molecule
H-2Dd on the resistant tumor cells because
P18-IIIB requires Dd to be presented to T cells.
As shown in Fig. 7a, resistant
tumor cells harvested from mice expressed Dd at
the same level as the original 15-12RM cells. Also, resistant tumor
cells could be lysed by P18 CTL when they were pulsed with P18-IIIB or
infected with vPE16 (Fig. 7b). These results showed that
class I expression on the resistant tumor cells is intact and that
resistant tumor cells can process and present the endogenously
expressed Ag normally. Therefore, we asked if they had lost expression
of the Ag. It was impossible to detect the expression of gp160 on the
15-12RM cells by Western blot analysis and flow cytometry. Thus, we
compared the expression of mRNA in resistant and nonresistant tumor
cells by RT-PCR. Fig. 8a shows
that mRNA for the gp160 v3 loop was absent in the resistant tumor
cells, while nonresistant tumor cells and the original 15-12RM cells
retained clear message. Next, we examined if this mRNA defect resulted
from the loss of the transfected gene or a mutation at the either of
the primer regions of v3 loop. DNA was isolated from 5 x
106 tumor cells and amplified by PCR. There were
the clear bands of amplified DNA by v3 loop primers for the resistant
tumors, as well as nonresistant tumors (Fig. 8b). These PCR
products were cloned and then sequenced from three resistant tumors and
two nonresistant tumors harvested from mice and the original 15-12 RM
cells. There was neither deletion of genes nor point mutations in the
v3 loop region (data not shown). These results suggested that the
tumors developed resistance against CTLs for P18-IIIB by decreasing the
expression of mRNA encoding the gp160 v3 loop.
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As mentioned earlier, the regrowth of tumor could be due either to
the development of escape variants of the tumor, or to loss of CTL
activity when the tumors reappear. Although the evidence above pointed
to the outgrowth of resistant variants of the tumor, we also wanted to
examine the possibility of CTL loss. Mice with large recurrent tumors
were examined on day 62 after tumor inoculation. Spleen cells were
restimulated for 6 days with the original tumor cells 15-12RM as
stimulators, and then tested in a lytic assay on both 15-12RM targets
and on 18Neo BALB/c 3T3 fibroblasts either with no peptide as a
negative control or pulsed with 1 µM peptide P18-IIIB (Fig. 9). As can be seen from the two mice
shown, a very vigorous CTL response was still present, specific for
both HIV-1 gp160 peptide and tumor cells, even after substantial
outgrowth of recurrent tumor. Thus, tolerance induction cannot account
for the observed outgrowth of tumor.
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Production of IFN- by CD8 cells contributes to protection
against the regrowth of tumors in CD4-depleted mice
As described above, depletion of CD4 cells, including Th cells,
led to the protection against regrowth of 15-12RM tumors after
regression. Since previous literature suggested that Th1 cells played
an important role in protection against viral infection and tumor
development (53, 54, 59), we utilized IFN- KO mice and IL-4 KO mice
as models of Th1/Th2 imbalance to examine the influence of these
cytokines on tumor growth and recurrence and on the prevention of
regrowth by anti-CD4 mAb injection. Though some of the IFN- KO
mice rejected tumors after initial growth, tumors regrew within 20 days
after the inoculation, statistically significantly more rapidly than
the control wild-type mice (Fig. 10b)
(p < 0.005, wild type vs IFN- KO, Log-Rank
test). On the other hand, surprisingly, IL-4 KO mice did not
significantly differ from control wild-type mice with regard to the
pattern of tumor development. They were not protected against regrowth
of tumors as observed with anti-CD4 treatment, even though they had
a skewed balance toward Th1 caused by the absence of IL-4 (Fig. 10c compared with Fig. 10a). When CD4 cells were
depleted before tumor inoculation, all the mice showed better
protection than mice injected with control rat IgG Ab (ICN
Pharmaceuticals) in each group (Fig. 10, a–c). The results
using IL-4 KO mice indicate that the complete loss of IL-4 and the
great diminution of other Th2 cytokines that are in part dependent on
IL-4 for their production (66, 67, 68, 69), does not mimic the effect of CD4
depletion and, therefore, does not explain the striking protection in
CD4-depleted mice. However, since tumors could regrow in CD4-depleted
IFN- KO mice (Fig. 10b), these results suggested that
IFN- produced by CD8 cells or NK cells played an important role in
protection against regrowth of tumors in CD4-depleted mice.
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a,
CD8+ T cells from CD4-depleted mice, without
restimulation in vitro, could kill 15-12RM target cells, as well as
18Neo pulsed with 1 µM of P18-IIIB, while CD8+
cells from tumor-inoculated, normal mice could not kill 15-12RM cells
without restimulation. Also, purified CD8 T cells from CD4-depleted
mice injected with tumor could lyse 18Neo pulsed with 1 µM of
P18-IIIB better than those from normal mice injected with tumor (about
15% vs 40% specific lysis at 80:1 E:T ratio). To further explore the
functional difference between these CTL, we examined the expression of
IFN- mRNA in these CD8+ T cells. Consistent
with the results of the CTL assay, we could detect four times as much
IFN- mRNA from CD8 T cells in CD4-depleted mice as from
CD8+ cells in tumor-inoculated untreated mice
(Fig. 11b). These results indicated that the quality of CD8
T cells in CD4-depleted mice was different from that in normal mice
with regard to IFN- mRNA expression, as well as CTL activity without
restimulation, properties that may be important for the protection
against recurrence of tumor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 9). Thus, this system serves as
a model of tumor immunosurveillance. In the clinical situation in
humans, many tumors express potential tumor Ags, and many subclinical
tumors may undergo this type of spontaneous regression, but only the
ones that escape immunosurveillance and reach the recurrent stage, as
in our model, are detected clinically. If we could understand what
prevents immunosurveillance from achieving complete regression and
allows tumor recurrence and later metastasis, we might be able to
overcome this obstacle and prevent tumor recurrence. This understanding
is also critical to designing optimal immunotherapy of cancer.
A potentially important clue to this problem came from the surprising
effect of CD4 depletion. Depletion of CD4 T cells did not change the
pattern of initial tumor growth and regression, but it inhibited
recurrence of the tumor. How could one explain the prevention of
recurrence of the tumor in CD4-depleted mice? In accordance with the
fact that CTL induction is associated with a Th-1 type immune response,
it has been shown that CD4 T cells that can secrete Th1-type cytokines
have a beneficial role in protection against tumor development (53, 54). Likewise, we previously reported that IL-2 production by human
peripheral lymphocytes in response to human papillomavirus Ags is
inversely associated with disease status (55). McAdam et al. (60)
showed that murine carcinoma cells transfected with IL-2 and IFN-
were more likely to be rejected than parental cells when implanted in
BALB/c mice. On the other hand, the shift from Th1-type to Th2-type
cytokine production was found in progressive cancer patients (61, 62),
and T cells harvested from tumor-bearing hosts produced only Th2-type
cytokines when they were stimulated in vitro (63). In addition to these
findings, Th2-type cytokines could even accelerate the experimental
pulmonary metastasis of melanoma (49).
Therefore, we hypothesized that the shift to a Th2-type response to
gp160 could be the cause of failure of complete tumor regression,
allowing recurrence of tumors after regression. We used IFN- KO mice
and IL-4 KO mice as a model of Th1/Th2 imbalance (64) to address this
question. Although the IFN- KO mice could suppress the initial
growth of tumor, these tumors finally developed earlier than those in
control BALB/c mice, suggesting a role for IFN-. However, other data
suggested a critical role for CD8 T cells: 1) vPE16-immunized mice that
have P18-IIIB-specific CTL before inoculation with tumor did not have
initial growth of tumors; 2) CD8-depleted mice could not inhibit tumor
growth at all; 3) Recurrent tumors had all become resistant to lysis by
CTL; and 4) We could induce a CTL response even from 15-12RM
tumor-bearing IFN- KO mice by stimulation with P18-IIIB in vitro
(data not shown). Thus, we concluded that both lytic activity by CD8 T
cells and production of IFN- are necessary for the regression of the
initial tumor in our model system.
Prior immunization with vaccinia virus vPE16, which induces
gp160-specific CTL, could prevent the initial growth of tumor
completely, but not its recurrence later. This result indicated that
the increase of CTL precursors for P18-IIIB contributed to clearance of
tumor cells, but could not eradicate them. In contrast, in CD4-depleted
mice, initial growth and regression of tumors were still observed but
the subsequent recurrence was prevented. Thus, the increase in CD8 T
cell numbers alone cannot explain the benefit of CD4 depletion. When
CD8 T cells were depleted by the injection of anti-CD8 mAb starting
from day 21 after inoculation of tumor cells in mice treated with
anti-CD4 mAb from the beginning, the tumors developed in only 25%
of these CD4-depleted mice (data not shown). This result indicated that
CD4-depleted mice might eliminate all of the tumor cells before the
critical point for recurrence, so that once the tumor cells were gone,
the CD8+ cells were no longer necessary. In
contrast, in the presence of CD4 cells, the clearance of tumor by CD8
cells was incomplete, even though CTL remained present during tumor
recurrence (Fig. 9).
However, unexpectedly, IL-4 KO mice did not mimic CD4-depleted mice in
that they could not stop the development of tumor after regression even
though they had a shift to Th1-type response in general (Fig. 10) (67, 65). IL-4 is necessary for the normal development of Th2 responses and
production of other Th2 cytokines (66, 67, 68), although some production of
IL-5 and IL-10 can still occur (69). The IL-4 KO mice thus indicate
that IL-4 is not required for the CD4-mediated prevention of complete
regression, and probably other Th2 cytokines, which are greatly
diminished in the IL-4 KO mice, also do not account completely for the
inhibitory effect of CD4 cells on the elimination of tumor. Therefore,
although a Th1-type response could contribute to the rejection of
initial tumors and a shift to a Th2-type response could interfere with
this protection, even a substantial skewing toward Th1-type response by
elimination of IL-4 was not sufficient to prevent recurrence of tumors
after regression. CD4 depletion must accomplish more than just Th2
depletion.
Koeppen et al. (70) observed that anti-CD4 treatment of mice
increased the frequency of rejection of an allogeneic tumor expressing
a foreign class I MHC molecule. Rakhmilevich and North (71) showed that
elimination of CD4 T cells augments the antitumor effect of IL-2
therapy in mice bearing an advanced sarcoma by releasing CD8 T
cell-mediated immunity from T cell-mediated suppression. Martinotti et
al. (72) reported that tumor infiltration by the CD8 T cells was
inhibited by CD4 T cells, but the tumors were unusual in being
transduced with the gene for IL-12. In that report, they postulated
that CD4-mediated suppression is exerted on CD8 expansion and on the
ability of CD8 T cells to infiltrate tumor nodules. However, we could
observe infiltration of lymphocytes in the tumor tissue not only in
CD4-depleted mice but also in untreated mice. Also, we found several
lines of evidence for a qualitative difference in CD8 cells from
CD4-depleted mice that appeared to contribute to more effective tumor
elimination. First, CD8 T cells purified from splenocytes of
CD4-depleted and tumor-injected mice had higher CTL activity specific
for 15-12RM cells than CD8 T cells from tumor-inoculated CD4-intact
mice, even without any stimulation in vitro. This result indicated that
CD8 T cells from CD4-depleted mice were already activated to kill tumor
cells efficiently in vivo. Second, this better lytic activity of CTL
was correlated with the expression of IFN- mRNA. Thus,
CD8+ T cells from CD4-depleted mice, which could
secrete more IFN-, could play an important role in prevention of
recurrence of tumor in our model system. We conclude that a qualitative
alteration of CD8 T cells following depletion of CD4 T cells could
account for protection from recurrence of tumor, whereas the reduction
in Th2-type cytokines in IL-4 KO mice was not sufficient.
We previously reported the importance of the quality of CTL as well as the quantity of CTL for viral clearance, in a study in which high-avidity CTL specific for P18-IIIB could protect better against virus challenge than low-avidity CTL (48). We are now investigating whether the quality of CTL has any correlation with high- or low-avidity CTL and whether there is any relation between CD4 depletion and the appearance of escape variant tumor cells.
Even during the recurrence phase of new tumor growth, a CTL response
was detected in spleen cells from these tumor-bearing mice after the
stimulation with P18-IIIB-pulsed spleen cells or 15-12RM cells (Fig. 9). This observation indicated that the exhaustion or tolerization of
responding cells against P18-IIIB was not the cause of tumor recurrence
after regression. There are several possible remaining explanations for
this escape mechanism of tumor cells from immunological destruction
(19). Besides the host factors that can suppress immune defenses
against tumor cells already discussed, there were several reports of Ag
loss of tumor variants (17, 18, 20). Selection of Ag loss, epitope
loss, or class I loss variants might lead to recurrence of tumor. In
this study, recurrent tumor cells became resistant (resistant tumor
cells) to CTL that could lyse both the original tumor cells and also
the tumor cells recovered from the initial growth stage before
regression (nonresistant tumor cells). The resistant tumor cells
remained class I-positive at the same level as the original tumor cells
by FACScan, and could process and present endogenous Ag, gp160, as
shown by their ability to be lysed by P18-IIIB-specific CTL when
infected with recombinant vaccinia virus expressing gp160. Since gp160
on 15-12RM cells could not be detected by Western blot and flow
cytometric analyses, we investigated the presence of DNA and the
expression of mRNA. We could detect amplified DNA of the v3 loop
(containing P18-IIIB) of gp160 from resistant tumors as well as from
the original 15-12RM and nonresistant tumors, and the DNA did not
contain any point mutation at any coding position within or near the v3
loop. However, only resistant tumor cells lost the expression of mRNA
for the v3 loop. Moreover, tumor cells recovered from CD8-depleted mice
could be lysed by CTL specific for P18-IIIB. Thus, this acquisition of
resistance against CTL occurred only in the presence of immune pressure
by CD8 T cells. These resistant tumor cells could grow in normal BALB/c
mice without regression and were not killed by CTL after several weeks
of culture in G418-containing medium, which means that escape variants
selected by CD8 T cells were stable both in vivo and in vitro, in
contrast to the situation in another report (73). Our results make
contamination of escape variants in the original 15-12RM cells
unlikely. In vivo, the tumor is removed from the G418-containing
selection medium used to select for neomycin-resistant cells. However,
this removal is unlikely to play a role in the escape for several
reasons. First, the original 15-12 cells were made by cotransfection
with NeoR and gp160 genes on
different plasmids, so that NeoR could be
used to select for cells that took up DNA in the original transfection,
but selective pressure from G418 should not affect retention of the
gp160 gene (38). Second, the original 15-12 transfectants continue to
express gp160 for at least 3 mo in culture without G418. Third, the
failure to develop resistant tumors in the CD8-depleted mice (in the
absence of G418), as just mentioned, implies that the selection for
resistance requires CD8-mediated immune pressure. Fourth, the gene for
gp160 is retained, but just the mRNA expression is lost. Thus, it is
very important to know how CTL pressure can cause the decrease of mRNA
expression of the epitope region in tumor cells to allow escape from
immunological surveillance.
In conclusion, this model has allowed us to begin to dissect some of
the mechanisms mediating and regulating tumor immunosurveillance.
CD8+ CTL appear to be critical for causing tumor
regression, but quantity of CTL alone is not sufficient. Rather,
qualitatively different CTL that produce more IFN- and remain
activated in vivo may be critical. The qualitative difference in CTL is
influenced by CD4+ cells that regulate the
CD8+ response, but this regulation cannot be
explained simply by the Th1/Th2 balance. Further studies to determine
the mechanism of this regulation will be important for designing
optimal immunotherapy. It may be valuable to induce CTL producing high
amounts of IFN-, as well as having high avidity, to obtain good
quality CTL for immunotherapy. Such CTL could be a useful component of
a strategy to prevent escape variants of tumor cells and to prevent
recurrence of tumors to control malignant disease. Concurrent
abrogation of the inhibitory effects of CD4 cells without eliminating
IFN- production may provide a successful concerted approach to
cancer immunotherapy.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: KO, knockout; HPRT, hypoxanthine-guanine phosphoribosyl transferase.
Received for publication January 27, 1999. Accepted for publication April 15, 1999.
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), 18Neo pulsed with 1 µM of
P18-IIIB (
), and 15-12RM cells (
).
),
harvested from vSC8-immunized mice at the same stage (x) and from
vPE16-immunized mouse (
) were used as target cells.
b, Tumor cells recovered during initial growth of tumors
from naive mice were lysed by 15-12RM CTL (• and 
).
c, Tumor cells from anti-CD8-treated mice were lysed
by the CTL line specific for 15-12RM (
) and
second round (
) or infected with vSC8 (
