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Int. J. Cancer: 70, 98–105 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
DENDRITIC EPIDERMAL T-CELL INVOLVEMENT IN INDUCTION
OF CD81 T CELL-MEDIATED IMMUNITY AGAINST AN
ULTRAVIOLET RADIATION-INDUCED SKIN TUMOR
Lois L. CAVANAGH1*, Ross StC. BARNETSON1, Antony BASTEN2 and Gary M. HALLIDAY1
of Medicine (Dermatology), University of Sydney at Royal Prince Alfred Hospital, Camperdown, Australia
2Centenary Institute of Cancer Medicine and Cell Biology, Camperdown, Australia
1Department
Murine epidermis contains 2 distinct cell populations which
contribute to the skin immune system, Langerhans cells (LC),
and dendritic epidermal T cells (DETC). LCs are important in
the induction of immunity against a wide range of antigens;
however, the function of DETC is unclear. To investigate the
roles of these epidermal cells (EC) in protective antitumor
immunity, an in vivo model of an ultraviolet radiation-induced
fibrosarcoma, UV-13-1, was used. Mice were immunized with
tumor antigen-pulsed EC followed 10 days later by an injection into the ear of 105 tumor cells, which did not lead to
formation of a detectable tumor, but was intended to simulate the influence of a developing tumor on the ensuing
immune response. The mice were then challenged with 2 3
106 viable tumor cells in each flank, sufficient to result in
growth of a measurable tumor. Protective immunity was
induced by DETC, and shown to be long-lasting, with tumors
inoculated 160 days after immunization being effectively
rejected. The effector cells responsible for protective immunity were CD81 T cells. Delayed-type hypersensitivity generated by tumor antigen-pulsed EC was dependent on LCs, with
no involvement of DETCs. This response, in contrast to that
of DETC, required prior culture of EC with GM-CSF, but
failed to inhibit tumor growth or incidence. Thus DETC and
LC can both activate antitumor immune responses, although
only the DETC-dependent response results in protective
immunity in the presence of a developing tumor. Int. J.
Cancer, 70:98–105, 1997.
r 1997 Wiley-Liss, Inc.
The immune system is thought to play a critical role in immune
surveillance against developing cutaneous tumors. The initiation of
immune responses against these tumors requires both the recognition of the tumor by local antigen-presenting cells (APC), and the
subsequent presentation of tumor-specific antigens to T lymphocytes (Elmets et al., 1995). Dendritic APC, which are the most
potent cells for induction of primary T cell-dependent immune
responses (Steinman et al., 1993), have been shown to be involved
in the induction of antitumor immunity (Grabbe et al., 1991).
However, the cellular events involved remain unknown, although
they are likely to be influenced by certain properties of the tumor
itself, including the stage at which it expresses tumor antigens, as
well as other factors such as the cytokines it may produce.
Most human skin tumors arise within the epidermis. Hence it is
reasonable to assume that epidermal cells (EC) will play a role in
the induction of skin cancers. Murine epidermis contains 2
populations of cells belonging to the immune system, i.e., dendritic
Langerhans cells (LC), and dendritic epidermal T cells (DETC),
which comprise 1–5% of total epidermal cells, depending on
mouse strain (Stingl et al., 1978; Sullivan et al., 1985). DETC
express CD3, Thy-1.2 and the gd T-cell receptor (Vg3/Cg1/Vd1)
for antigen (Kuziel et al., 1987). The function of these cells has not
been fully established; however, in vitro studies have shown that
DETC cell lines, unlike splenic T cells, can lyse a range of
skin-tumor cell lines, but not normal keratinocytes (Kaminski et
al., 1993). Moreover, when admixed with the melanoma cell line
K1735, DETC have been shown to prevent tumor growth after
intradermal injection (Love-Schimenti and Kripke, 1994b). By
contrast, other studies have suggested that DETC exert inhibitory
effects on the growth of cultured tumor cells (Welsh et al., 1992;
Love-Schimenti and Kripke, 1994a).
LC, which function as professional antigen-presenting cells,
have been shown to be important in induction of contact hypersensitivity and delayed-type hypersensitivity (DTH) (Stingl et al.,
1978; Sullivan et al., 1986). There has been much speculation as to
their role in antitumor immunity, although LC have been shown to
be involved in induction of protective immunity against a chemically-induced tumor (Grabbe et al., 1991).
As ultraviolet (UV) radiation is the major cause of skin cancers
in humans (Dubin et al., 1990), and the immunogenicity, tumor
antigens and biological behavior of UV-induced tumors is likely to
be different than in tumors induced by other carcinogenic agents
(Prehn and Main, 1957; Kripke, 1974), it is important to determine
the part played by the immunocompetent cells of the epidermis in
initiation of immune responses against such tumors. In an attempt
to simulate the effects exerted by a tumor during the early stages of
development, we immunized mice with tumor antigen-pulsed EC
followed by a dose of tumor cells insufficient to form a detectable
tumor, but sufficient to elicit a DTH response. Protective antitumor
immunity was then assessed by inoculating the mice with a large
enough number of tumor cells to produce a growing tumor. The
tumor selected for this purpose was the UV-induced fibrosarcoma,
UV-13-1, which grows progressively in immunocompetent mice,
thus enabling the investigation of immunity which protects against
tumor growth.
MATERIAL AND METHODS
Mice
Inbred male and female mice of the C3H/HeNCr strain were
between 8–12 weeks old at the beginning of the experiments. Mice
were used in accordance with University of Sydney Animal
Experimental Ethics Committee guidelines.
Tumor cell line
The UV-induced fibrosarcoma UV-13-1, originally derived in a
C3H/HeNCr mouse, was kindly provided by Dr. M.L. Kripke,
M.D. Anderson Cancer Center (TX). Both the tumor and mice were
tested periodically by isoenzyme analysis to ensure genetic drift
had not occurred. The UV-13-1 tumor line was maintained in tissue
culture at 37°C and 5% CO2 in Dulbecco’s modification of Eagle’s
media (DMEM; Trace Biosciences, Castle Hill, Australia) containing 10% newborn calf serum (NCS; CSL, Parkville, Australia), 20
mM HEPES buffer and 8 mM L-glutamine. UV-13-1 grows
progressively when injected subcutaneously or intradermally in
immunocompetent syngeneic recipients.
*Correspondance to: University of Queensland Department of Medicine,
Princess Alexandra Hospital, Woolloongabba, Queensland 4102, Australia.
Fax: 617 3240 5399. E-mail: loisc@gpo.pa.uq.edu.au
Abbreviations: DETC, dendritic epidermal T cells; DTH, delayed-type
hypersensitivity; EC, epidermal cells; LC, Langerhans cells; TE, tumor
extract; UV, ultraviolet.
Received 17 June 1996; revised 14 September 1996.
ANTITUMOR IMMUNITY AND DENDRITIC EPIDERMAL T CELLS
Preparation of tumor extract (TE)
UV-13-1 tumors were grown in mice which had been immunosuppressed by prior exposure to an acute dose of UV radiation over
5 days, giving a total dose of 6.82 J/cm2 UVA, and 0.59 J/cm2 UVB.
The tumors were excised when approximately 10–15 mm in
diameter, placed into sterile PBS, disaggregated mechanically and
then sonicated to disrupt cell membranes. The supernatant, collected after centrifugation at 1,500g for 30 min at 4°C, was
dialyzed against PBS (12–14 kDa cutoff, SpectraPor Membrane
Tubing, Spectrum Medical Industries, Houston, TX), and used as
99
the source of tumor antigen. Protein concentration was determined
by the Lowry method.
Preparation of epidermal cells (EC)
EC were prepared according to a modification of a method
previously described (Sullivan et al., 1985). Briefly, mice were
sacrificed, and the dorsal trunk shaved and chemically depilated
(Veet, Reckitt and Coleman, West Ryde, Australia). The excised
skin was cut into small pieces, placed into Ca21/Mg21 free-HBSS
supplemented with 20 mM HEPES (HEPES-BSS) containing 0.3%
trypsin (Boehringer-Mannheim, Germany) and 300 U/ml DNase
(Amersham, Takara, Japan) and incubated for 16 hr at 4°C. The
epidermis was mechanically separated from the underlying dermis,
and reincubated in fresh 0.3% trypsin solution containing 300 U/ml
DNase for 20 min at 37°C, after which an equal volume of DMEM
containing 10% NCS was added. EC were dislodged by gentle
agitation, filtered through 250-µm nylon mesh (Swiss Screens,
Seven Hills, Australia) and washed twice.
Following preparation, EC were incubated for 2 hr at 37°C in
DMEM containing 10% NCS, 20 mM HEPES, 8 mM L-glutamine
and 0.05 mM 2-mercaptoethanol (complete medium) prior to use.
In some experiments, 10 U/ml murine recombinant granulocytemacrophage colony-stimulating factor (GM-CSF; Genzyme, Cambridge, MA) were added during this culture.
In vitro depletion of EC subsets
MHC class II1 EC were removed prior to the 2-hr culture by
incubation with monoclonal anti-Ia antibody (TIB 93; ATCC,
Rockville, MD) in the form of hybridoma supernatant diluted 1:4 in
complete medium, for 30 min at 4°C. Alternatively, Thy-11 EC
were removed by incubation in purified monoclonal anti-Thy-1.2
antibody (T-24-31.7; Dennert et al., 1980) diluted 1:500 in
complete medium. The EC were washed and incubated at 37°C for
35 min in low-toxicity rabbit complement (Cedarlane, Hornby,
Canada) diluted 1:30 in PBS containing 5% fetal calf serum (FCS)
(CSL, Parkville, Australia). Dead cells were removed by treatment
with 0.05% trypsin and 300 U/ml DNase in HEPES-BSS for 10
min at 37°C, and then washed. Depletion of class II1 or Thy-11
cells was confirmed by immunofluorescence.
Antigen pulsing of EC and immunization protocol
EC were washed twice with HEPES-BSS containing 10% NCS,
and once with HEPES-BSS prior to exposure to TE at a concentration of 1,000 µg/ml in HEPES-BSS, for 30 min at 37°C. They were
then washed as before to remove any remaining TE, and resuspended for immunization in HEPES-BSS. Mice were immunized
with 104 viable TE-pulsed EC by s.c. injection into the dorsal trunk.
A sample of the EC pulsed with TE were killed by freeze-thawing
for use as nonviable controls; microscopic examination ensured
that no intact cells remained. Ten days following immunization
with TE-pulsed EC, mice were injected with 105 viable UV-13-1
cells in PBS in the dorsal pinna of one ear; this was a dose of tumor
FIGURE 1 – Antitumor protective immunity is dependent on immunization with TE-pulsed EC and a low dose of tumor cells. EC cultured in
10 U/ml GM-CSF for 2 hr were pulsed with TE and then injected s.c.
into 2 groups of mice (104 EC/mouse). One group was injected with 105
UV-13-1 tumor cells into the ear 10 days later (group a) and the DTH
induced by EC measured. Another group, given only tumor cells,
served as control (group c). (a) DTH response (mean ear swelling
response 6 SEM) was measured 24 hr later. These groups were then
inoculated with 2 3 106 UV-13-1 cells i.d. into each flank 7 days later.
Mice immunized with TE-pulsed EC only (group b) and naive mice
(group d) were inoculated with tumor cells 10 days after immunization
with EC. (b) Tumor growth (mean tumor diameter) was measured over
the 28 days following tumor inoculation (p , 0.001, least significant
difference 5 2.28). (c) The incidence of tumors (% mice per group
with tumors) was determined at each time point. All mice were
compared with the naive control group. *p , 0.05; n 5 7/group.
100
CAVANAGH ET AL.
cells insufficient to form a tumor (subsequently referred to as
‘‘tumor cell immunization’’).
Assessment of delayed-type hypersensitivity
DTH responses induced by TE-pulsed-EC were determined 24
hr after tumor-cell immunization as the difference in thickness
between the injected and uninjected ears, measured using an
engineer’s micrometer. A control group of mice which received
tumor-cell immunization without EC was included in each experiment.
Assessment of protective antitumor immunity
Protective antitumor immunity was assessed by both incidence
and growth of subsequently inoculated tumor cells. Mice immunized with TE-pulsed EC plus tumor cells, or those given the
tumor-cell immunization only, received an intradermal inoculation
of 2 3 106 UV-13-1 live tumor cells in 50 µl PBS into each flank
between 17–25 days after exposure to TE-pulsed EC. The development of tumors was assessed by manual palpitation 7 days after
inoculation of tumors, and then every 3–7 days until the end of the
experiment (4–5 weeks). In those mice which developed tumors,
tumor diameter was measured with vernier calipers. In all experiments, a naive group of unimmunized mice was included, which
received tumor inoculation at the same time as immunized mice.
In vivo depletion of T-cell subsets
Mice were depleted of T-cell subsets according to the protocol
described by Cobbold et al. (1984). Briefly, mice were injected
intraperitoneally with the optimal concentration of purified antibody (Ab), previously determined to result in maximal depletion of
T-cell subsets, on 3 consecutive days, and then at weekly intervals
throughout the experiment. Antibodies included were anti-CD4
(GK1.5; ATCC), and anti-CD8 (YTS169.4; Cobbold et al., 1984)
used at 1.0 and 4.0 mg protein per injection, respectively. Anti-CD4
Ab removed approximately 80% of splenic CD41 T cells, and
anti-CD8 Ab removed approximately 75% of CD81 T cells. The
anti-mycobacteria Ab (L22, 1.0 mg protein per injection; a kind gift
from Dr. W. Britton, Centenary Institute of Cancer Medicine and
Cell Biology, Camperdown, Australia), used as a control, did not
alter either CD8 or CD4 T-cell subpopulations.
Statistical evaluation
DTH results were analyzed using the unpaired Student’s t-test,
the results from all groups being compared to the control group
within each experiment. Significance was determined at 95%
confidence limits (p , 0.05). Tumor growth measurements were
taken every 3–7 days after tumor inoculation until the end of the
experiment, and the average size of tumors per mouse at each time
point over the entire length of the experiment were included in the
statistical analyses. This was done by multivariate analysis of
variance (ANOVA), taking into account all individual tumor
measurements throughout the duration of the experiment. Results
are presented as mean tumor diameter of the 2 tumors possible at
each time point. Fisher’s protected least significant difference was
used to test for significant differences between groups within the
analysis. As the incidence data indicate the number of mice without
tumors, any tumors which had been rejected were excluded from
the growth curve. The incidence of tumors which was determined
throughout the experiment was analyzed using the Mann-Whitney
U test for nonparametric analysis. Significance was determined at
95% (p , 0.05) for both tumor growth and incidence.
FIGURE 2 – Live EC induce protective antitumor immunity. EC were
cultured with (groups a and c) or without (groups b and d) 10 U/ml
GM-CSF for 2 hr prior to pulsing with TE. Viable EC were injected s.c.
immediately (groups a and b); a sample of EC was killed by
freeze-thawing before injection (groups c and d). These mice, and a
group which did not receive EC (group e), were reimmunized with 105
tumor cells into the ear 10 days after immunization with EC. (a) DTH
(mean ear swelling response 6 SEM) elicited by tumor cells was
measured 24 hr later. All mice were compared with control group e. All
groups of mice, plus naive, unimmunized group f, were inoculated with
2 3 106 UV-13-1 tumor cells i.d. into each flank 7 days later. (b) Tumor
growth (mean tumor diameter) was measured throughout the duration
of the experiment (p , 0.01, least significant difference 5 1.97). (c)
The incidence (% mice per group with tumors) of tumors was
determined at each time point. All groups were compared with the
naive, unimmunized control mice (group f). *p , 0.05; n 5 6/group.
ANTITUMOR IMMUNITY AND DENDRITIC EPIDERMAL T CELLS
RESULTS
Protective anti-tumor immunity is dependent upon immunization
with both TE-pulsed EC and tumor cells
Two groups of mice (a and b) were immunized with 104 EC
pulsed in vitro with UV-13-1 TE. One group (a) was given 105
viable UV-13-1 tumor cells into the pinna of one ear 10 days later.
This and a tumor-cell-immunized control group (c) were inoculated
with 2 3 106 UV-13-1 tumor cells s.c. into each flank after 7 days.
The other EC-immunized group (b) and a naive control group (d)
101
were also inoculated with UV-13-1 tumor cells 10 days after
immunization with EC. DTH was assessed 24 hr after immunization with tumor cells. Immunization of mice with TE-pulsed EC
induced a DTH response significantly greater than that of the
control (Fig. 1A).
Immunization with TE-pulsed EC and a low dose of tumor cells
induced significant protective immunity against subsequent inoculation of UV-13-1 tumor cells, illustrated by a significantly reduced
tumor growth rate compared to those tumors growing in naive mice
(Fig. 1B). Protection was dependent upon immunization with both
TE-pulsed EC and tumor cells, as neither EC immunized mice (b)
nor mice given only a low dose of tumor cells (c) demonstrated
significantly reduced tumor growth compared with naive mice (d).
Immunization with TE-pulsed EC and tumor cells (a) also induced
significant tumor rejection compared to naive control mice (Fig. 1C).
However neither cell population alone affected tumor incidence.
Live EC induce protective antitumor immunity
Prior to pulsing with TE, EC were cultured for 2 hr either with
(groups a and c) or without (groups b and d) 10 U/ml GM-CSF.
Mice were immunized with these viable EC (a and b) or with EC
killed by freeze-thawing (c and d). These 4 groups of mice, and a
further group (e), were injected in the ear with 105 live UV-13-1
tumor cells 10 days later. When DTH was measured 24 hr later,
only viable EC cultured with GM-CSF induced a significant
response, whereas nonviable EC, or EC cultured without GM-CSF,
did not (Fig. 2A). These groups, plus a naive control group (f), were
then inoculated with 2 3 106 tumor cells 7 days later. Mice immunized with viable EC cultured with GM-CSF and tumor cells (a)
rejected all tumors by 3 weeks (Fig. 2C). Immunization with viable
EC cultured without GM-CSF and tumor cells (b) also exerted
significant protective effects. Tumor growth (Fig. 2B) was significantly less than the growth of tumors in naive mice (f), and tumor
incidence was significantly reduced to 20% by 4 weeks. Immunization with nonviable EC (c and d) did not alter tumor growth or
incidence, nor did tumor cells in the absence of EC immunization
(e). Thus, viable EC were required for the induction of protective
immunity. Using multiple immunizations, however, we found that
viable EC were not required for the induction of protective
immunity. Viable EC were necessary for the induction of DTH, as
this aspect of immunity was totally dependent on EC viability (data
not shown). Culture of EC with GM-CSF was necessary for induction
of DTH. However, GM-CSF was not required for protective antitumor
immunity, although it did slightly enhance antitumor immunity in some
experiments. In all subsequent experiments presented, EC were cultured
for 2 hr with 10 U/ml GM-CSF prior to pulsing with TE.
Protective antitumor immunity is long-lived
To investigate whether the antitumor immunity induced by
TE-pulsed EC is long-lived, mice were immunized with either
viable or nonviable EC pulsed with TE, followed by reimmunization with 105 tumor cells in the ear, as in previous experiments.
Once again, viable but not killed EC induced a significant
antitumor DTH (Fig. 3A). The mice then received 2 3 106 tumor
cells, approximately 5 months (160 days) after immunization.
FIGURE 3 – Antitumor immunity induced by immunization with EC
is long-lived. Mice were immunized s.c. with either 104 viable (group
a) or killed (group b) EC pulsed with TE. These mice and an untreated
group were given 105 UV-13-1 tumor cells into the ear 10 days later. (a)
DTH response (mean ear swelling response 6 SEM) was assessed at
24 hr. Immunized mice were compared with control group c. All
immunized, and a group of naive, unimmunized mice (group d), were
inoculated with 2 3 106 tumor cells i.d. in each flank 160 days after EC
immunization. (b) Tumor growth (mean tumor diameter) was measured
throughout the experiment ( p , 0.01, least significant difference 5 1.15). (c) Tumor incidence (% mice per group with tumors)
was determined at each time point. EC-immunized mice were compared with naive control group d. *p , 0.05; n 5 7/group.
102
CAVANAGH ET AL.
Compared with naive mice, the tumors in mice immunized with
viable EC and tumor cells displayed a reduction in both growth and
incidence (Fig. 3B, C). On the other hand, immunization with
killed EC and tumor cells did not affect growth, although it did lead
to reduced incidence. However, the reduction in incidence was not
as great as that in mice immunized with viable EC (Fig. 3C). This
demonstrates that the protective antitumor immunity induced by
viable EC pulsed with TE, followed by a low dose of tumor cells, is
long-lived and capable of inducing tumor rejection at least 5
months after immunization.
Induction of antitumor immunity by EC subsets
Prior to culture with GM-CSF, class II (Ia)1 or Thy-11 EC were
depleted by monoclonal antibody-mediated complement lysis.
Class II1 EC were depleted from approximately 4% to 1.3%, and
Thy-11 EC from approximately 4% to 0.5%, as determined by
immunofluorescence. These subset-depleted EC, and unfractionated EC, were pulsed with TE, and used to immunize mice. These
and unimmunized mice were injected in the ear with 105 UV-13-1
tumor cells. On measuring ear thicknesses 24 hr later, it was found
that the class II1 EC were responsible for induction of DTH against
the UV-13-1 tumor, as class II1-depleted EC were unable to induce
this response, whereas the groups given unfractionated, or Thy-1depleted EC, had DTH responses significantly higher than the control
(Fig. 4A). Hence, class II1 EC were responsible for the induction of
DTH against the UV-13-1 tumor, whereas Thy-11 EC were not.
Mice immunized with EC and tumor cells, plus groups of naive
mice, were injected intradermally with 2 3 106 UV-13-1 tumor
cells in each flank. In 3 of 4 repeat experiments, it was found that
depletion of class II1 EC (group b; Fig. 4B) had no effect on the
incidence of tumors compared with mice immunized with unfractionated EC (group a). The tumor incidence in both these groups
was significantly reduced compared with naive control group c.
Hence, class II1 EC are not responsible for inducing protective
antitumor immunity against the UV-13-1 tumor, although this cell
type did induce DTH against the same tumor. However, depletion
of Thy-11 EC did affect tumor incidence (Fig. 4C). Mice immunized with unfractionated EC (group d) again showed marked
reduction in tumor incidence compared to the naive control mice
(group f), whereas Thy-1 depletion (group e) prevented the
rejection of tumors. Hence, although uninvolved in the induction of
DTH against the UV-13-1 tumor, Thy-11 EC were responsible for
inducing the rejection of this tumor in immunized mice.
CD81 T cells are required for protective antitumor immunity
The role of T lymphocytes in antitumor immunity was investigated by depleting the mice of T-cell subsets by repeated injections
of anti-CD4, anti-CD8 or a control antibody at the time of
inoculation with 2 3 106 viable UV-13-1 tumor cells. The
injections of Ab continued throughout each experiment to ensure
adequate depletion of T cells. Treatment with anti-CD8 Ab resulted
in a reduction of approximately 71% of splenic CD81 T cells, with
no effect on CD41 T cells, and anti-CD4 Ab removed approximately 70% of splenic CD41 T cells without affecting CD8 cells.
Treatment with the control Ab did not influence either T-cell subset.
The growth of tumors in T-cell-depleted mice was compared with
control mice, which were not treated with Ab. In unimmunized
mice, depletion of CD81 T cells significantly enhanced tumor
growth, whereas depletion of CD41 T cells or use of the control Ab
had no effect. On the other hand, there was no difference in the
incidence of tumors in any group (data not shown).
The next step was to investigate the effects of T-cell subset
depletion in mice immunized with TE-pulsed EC given 10 days
before injection of 105 tumor cells into the ear. This form of tumor
cell immunization elicited the expected antitumor DTH response in
all EC-immunized mice (Fig. 5A). Seven days later, the mice were
inoculated with 2 3 106 viable tumor cells in the flank, and at the
same time treatment with purified antibodies was commenced. In
mice injected with the control Ab (group a), tumor growth was
significantly reduced compared with that in naive unimmunized
FIGURE 4 – Role of class II1 and Thy-11 EC in induction of
antitumor immunity. Mice were immunized s.c. with 104 TE-pulsed EC
or EC depleted of class II1 or Thy-11 cells by Ab-mediated complement lysis. These mice and an unimmunized group were given 105
tumor cells into the ear 10 days later, and DTH were assessed at 24 hr.
(a) DTH (mean ear swelling response 6 SEM) responses induced by
immunization were compared with control mice. Mice immunized with
unfractionated EC (groups a and d), class II-depleted (group b), or
Thy-1-depleted (group e) EC, together with naive, unimmunized mice
(groups c and f), were inoculated with 2 3 106 tumor cells i.d. in each
flank 7 days later. (b,c) Tumor incidence (% mice per group with
tumors) was determined at each time point. Immunized mice were
compared with naive, unimmunized mice. *p , 0.05; n 5 7/group.
ANTITUMOR IMMUNITY AND DENDRITIC EPIDERMAL T CELLS
mice (group d; Fig. 5B). This effect persisted following depletion
of CD41 cells (group c). However, tumor growth in mice depleted
of CD81 lymphocytes (group b) was comparable to that of the
naive group. The incidence of tumors in EC-immunized mice given
control Ab was significantly reduced compared to the naive control
group, as was the incidence in CD41 T-cell-depleted mice (Fig.
5C). By contrast, depletion of CD81 T cells completely abolished
protective immunity induced by immunization with EC and tumor
cells. In other words, CD81 but not CD41 effector cells are
required for protective immunity in this system.
103
DISCUSSION
The studies reported here made use of a model in which
immunization with tumor antigen-pulsed EC was followed by
injection of a low number of viable tumor cells in an attempt to
simulate induction of antitumor immunity during the early stages
of a developing tumor. The protective effect of this immunization
protocol was then determined by challenging the mice with a
sufficient number of tumor cells to form a detectable tumor. The
results pointed to involvement of distinct EC populations in
induction of DTH and protective immunity, with gd1 DETC
playing a role in protection, and with MHC class II1 LC in DTH. In
other words, 2 different epidermal cell subpopulations were
responsible for the 2 different types of antitumor immunity
observed.
Induction of protective antitumor immunity depended on both
DETC and live tumor cells. Neither immunization with a single
injection of TE pulsed-EC nor a low dose of tumor cells alone
initiated a protective response. Moreover, a second injection of EC
pulsed with tumor extract failed to substitute for viable tumor cells
(data not shown). Thus, induction of protective immunity in this
system appears to be dependent on 2 separate and different steps,
the first mediated by DETC and the second by tumor cells per se.
The part played by DETC in cutaneous immunity in vivo is not
clearly understood. Here we have presented in vivo evidence of a
role for DETC in protective antitumor immunity. DETC cell lines
have been shown to influence the growth of tumor cells in vitro, by
directly lysing a range of tumor cell lines (Kaminski et al., 1993).
Based on this evidence it has been postulated that DETC may play
a role in host resistance to neoplastic cells. An in vivo study has
shown that coinjection of cultured DETC and K1735 melanoma
cells abrogates normal growth of these tumors (Love-Schimenti
and Kripke, 1994b), although if the DETC and tumor cells were
injected simultaneously at different sites there was no inhibitory
effect on tumor growth, indicating a direct effect of the DETC on
tumor cells. By contrast, in our experiments the initial subcutaneous injection of EC, containing DETC, was followed by a low dose
of tumor cells given intradermally at a remote site 10 days later.
Thus, DETC do not appear to be functioning in a similar way in our
system.
CD81 T lymphocytes were involved in rejection of the UV-13-1
tumor in EC-immunized mice. Depletion of CD81 T cells in
unimmunized mice also led to an accelerated growth rate, indicating that there was an immune response generated by the tumor
itself which limited growth without inducing tumor rejection.
These results are in agreement with previous studies demonstrating
a role for CD81 T cells in limiting the growth of UV-induced
tumors (Fortner and Kripke, 1977). On the other hand, CD81 T
cells are not present in DETC (Bergstresser et al., 1983), which
means that the protective effect of DETC may be mediated by
activation of CD81 T cells. Activation of CD81 T cells depends on
presentation of antigens in association with MHC class I, for
example by bone-marrow-derived, nonprofessional antigen-
FIGURE 5 – Antitumor immunity induced by TE-pulsed EC and
tumor cells in mice depleted of T-cell subsets. Mice were immunized
s.c. with EC pulsed with TE, and then these and another group were
given 105 tumor cells into the ear 10 days later. (a) DTH response
(mean ear swelling response 6 SEM) was measured 24 hr later, and
EC-immunized mice were compared with mice immunized with tumor
cells only. These mice, and naive group d, were inoculated with 2 3 106
tumor cells i.d. in each flank 7 days later. At the same time, the
EC-immunized mice were divided into 3 groups, one being injected
repeatedly with anti-mycobacteria Ab (group a), the second injected
with anti-CD8 Ab (group b), and the third with anti-CD4 Ab (group c).
(b) Tumor growth (mean tumor diameter) was measured over the next
35 days (p , 0.001, least significant difference 5 2.30). (c) Tumor
incidence (% mice per group with tumors) was determined at each time
point. All EC-immunized groups were compared with naive, unimmunized control mice (group d). *p , 0.05; n 5 7/group.
104
CAVANAGH ET AL.
presenting cells in the case of B16 melanoma (Huang et al., 1994).
This implies that tumor antigens can be reprocessed by the host’s
APC after immunization. However, this does not appear to apply to
our model, since neither injection of DETC (tumor extract pulsedEC), nor a low dose of tumor cells alone, was sufficient to induce
protective immunity.
CD41 T lymphocytes may also be involved in the immunity
induced by DETC. When CD41 T cells were depleted in the
immunized animals, the immune response against the transplanted
tumor cells was enhanced, as all tumors were rejected by 14 days.
Although we did not observe such an absolute response in 3 other
similar experiments, a subsequent reduction in tumor growth still
occurred. Hence the data suggest a potentially suppressive role for
CD41 T cells in antitumor immunity generated by DETC. Moreover, in vivo depletion of CD41 T cells caused regression of a
tumor which required CD81 cytolytic T lymphocytes for rejection
(Koeppen et al., 1993). By contrast, Kosugi et al. (1987) have
shown that depletion of CD41 T cells diminished the CD81
T-cell-dependent response to X5563 plasmacytoma, an effect
which could be reversed by addition of T-cell-derived cytokines. In
our study CD41 T cells were not depleted until mice were
inoculated with tumor cells to assess tumor growth; thus, a role for
CD41 cells in providing help to CD81 effector cells early in the
antitumor immune response could not be excluded. Indeed, they
may well be the explanation for the result observed by Kosugi et al.
(1987).
Protective antitumor immunity generated by immunization with
DETC followed by tumor cells is still as effective 160 days after the
immunization as at 7 days, demonstrating that the protective effect
of DETC is long-lived. Previous studies using EC or splenic APC
to induce protective immunity have not investigated the longevity
of the antitumor immune response (Grabbe et al., 1991; Flamand et
al., 1994; Shimizu et al., 1989). Immunization against the UVinduced melanoma K1735, using tumor cells transfected with the
costimulatory molecule B7, revealed a discrete window of responsiveness following immunization, as protective immunity was
observed only when tumor cells were transplanted 25 days after
immunization, and not at 5 or 90 days (Townsend et al., 1994).
However, when the same immunization protocol was used for the
non-UV-induced EL-4 thymoma, protective immunity was observed up to 92 days postimmunization, the latest time tested in that
study. It is possible that in the case of our immunization protocol,
the low dose of tumor cells used for secondary immunization
persisted without developing into a tumor, thus providing a
continual source of tumor antigen.
LC have long been known to be integral to induction of
cutaneous immunity against a wide variety of antigens (Stingl et
al., 1978), and here we have confirmed their role in initiation of
antitumor DTH. The DTH response elicited by the exposure to a
low dose of tumor cells was dependent on immunization with LC,
not DETC, with only a single injection of live LC being required
provided they had been precultured with GM-CSF. By contrast, the
capacity of DETC to induce protective antitumor immunity was
unaffected by culture with GM-CSF, consistent with the findings
that protection and DTH were initiated by 2 different cell types in
this model.
LC have been shown to induce protective immunity against the
chemically-induced S1509a fibrosarcoma (Grabbe et al., 1991),
providing valuable information on the range of mechanisms by
which protective antitumor immunity can be induced. The EC used
in this study were depleted of DETC, as the presence of these cells
prevented induction of protective immunity, possibly by exerting a
suppressive effect on the antitumor response. The reasons for the
difference in antitumor mechanisms in this, and in our system, may
be due to the nature of the tumors used. Thus, tumors induced by
UV radiation express unique antigens and do not lead to crossprotective immunity against other tumors, irrespective of their origin
(Kripke, 1974), whereas some non-UV-induced tumors may induce
crossreactive responses (Prehn and Main, 1957). In addition, the
repertoire of cytokines produced by each tumor, or by the host in
response to a developing tumor, may influence the mechanism
responsible for immunity. Different cytokines have been shown to
influence tumor growth in distinct ways, and can broadly be
divided into those which are tumor-inhibitory, or tumor-promoting
(reviewed by Schreiber, 1993). The UV-13-1 tumor used in these
studies produces high levels of GM-CSF but low levels of TNFa
and no detectable IL-3 compared with other UV-induced murine
skin tumor lines which we have examined (D. Rubel and G.
Halliday, personal communication). Hence the nature of immune
responses, and the mechanisms by which immunity is induced
against tumors, can be affected by the tumor itself.
It is therefore likely that a variety of immunological mechanisms
can be activated by different EC populations, and that the tumor
itself can play a role in controlling the development of the
responses involved in protective anti-tumor immunity.
ACKNOWLEDGEMENTS
We thank Dr. G. Berry, Department of Public Health, University
of Sydney, for statistical advice, and Mr. R. Sluyter for technical
assistance and helpful discussions. The support of the National
Health and Medical Research Council, and of the University of
Sydney Cancer Research Fund, is also gratefully acknowledged.
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