Tumor antigen presentation by epidermal antigen-presenting cells in the mouse: modulation by granulocyte-macrophage colony-stimulating factor, tumor ...
Tumor cells
antigen
presentation
in the mouse:
colony-stimulating ultraviolet
by epidermal
modulation factor,
antigen-presenting
by granulocyte-macrophage
tumor
necrosis
factor
radiation
Stephan Richard
Grabbe Sandra D. Granstein
MGH-Harvard
Cutaneous and
Massachusetts,
Abstract: I-At epidermal APCs, Langerhans cells) tumor-associated antigens immunity in vivo. This
(
Bruvers,
Biology
*Department
Ann
Research
M. Lindgren,
Center,
of Dermatology,
Department University
antigen-presenting cells have been shown to present (TAAs) and to induce tumor study examined the effects of
Junichi
and
(GM-CSF)-cultured ble of presenting induction of in
the effects induction
of the cytokines or elicitation
209-217;
1992. Words:
reduced it in a dose-dependent these results demonstrate UVR are significant regulators by epidermal APCs and examined of immunity.
differ
J.
with
Leukoc.
regard Biol.
immunity
skin
cancer
cytokinet
Since
cutaneous that
tumor
are
cutaneous
that of that to 52:
malignancies.
tricted
fashion
in ref. involved
immunity,
thought
I-Ak
3), litin in-
for
to be
Langerhans
which
of crucial cells
(LCs)
In
this
context,
we
murine I-At epidermal tumor-associated antigens vivo tumor immunity in [7],
recently
showed
colony-stimulating
allowing
us
to
study
factor
cells are capa(TAAs) for the a genetically res-
factors
that
regulate
(
14), as
and
protein
mRNA
for
also is
for
IL-i,
IL-i,
IL-6,
IL-6,
been
found
in
possible
that
the
and
TNF-a
GM-CSF,
normal
[15-18]
and
epidermis
TNF-a
[19,
vivo.
Therefore,
capacity
of
in
antigen-presenting
as
well
20]
has it
resident
epidermal APCs is also influenced by these cytokines, among which GM-CSF and TNF-a appear to be the most relevant. Indeed, GM-CSF has been shown to prolong survival of LCs in culture, increase major histocompatibility complex (MHC) class II expression, and differentially modulate other surface
molecules
on murine
epidermal
dition, murme epidermal LCs present alloantigen to unprimed and either retain or down-regulate to
primed
T
LCs
[8, 9, 21].
In ad-
develop enhanced ability T cells during culture antigen processing
cells,
depending
on
the
to [22] and
mouse
Langerhans Abbreviations:
FCS, cell;
fetal
in immunosuppressed patients experimental data demonstrate immunity against spontaneous
3; by of
ety
Journal
for
Stephan
Cutaneous January
of this
work
Investigative
of Leukocyte
IL-i,
interleukin-l;
s.c.,
subcutaneously;
fragment; factor
Biology 31,
1992;
have
been
Dermatology,
Biology
delayed-type
hyper-
cell
granulocyte-macrophage
saline;
tumor
DTH,
fluorescence-activated
interferon--y,
necrosis
requests:
cell;
FACS,
GM-CSF,
IFN--y,
TF,
tumor
Reprint
Parts
serum;
antigen;
TNF-a,
Received
calf
cell;
phosphate-buffered
MGH-East, MA 02129.
[1, the
antigen-presenting
epidermal
factor;
PBS,
associated
mechanisms its support incidence
APC,
EC,
stimulating
INTRODUCTION
cutaneous malignancies 2]. Although abundant presence of T cell-mediated
specific
(APCs)
granulocyte-macrophage
sensitivity;
surveillance despite an increased
Boston,
cutaneous tumor immunity. The antigen-presenting function of several types of APCs are modulated by cytokines, such as interleukin-i (IL-i), IL-6, interferon-”y (IFN-”y), transforming growth factor f3 TGF-3), tumor necrosis factor a (TNF-a), and colonystimulating factors [8-13]. Keratinocytes are capable of producing most of these cytokines in vitro (reviewed in ref.
cells
The existence of tumor immune within the skin is still controversial, numerous clinical studies showing
School,
have been shown to present haptens and protein antigens for the generation of CD4-dependent immunity [4-7], our aim was to evaluate their role in defense mechanisms against
presentation
tumor
of
cells
vals with ECs that had been cultured in GM-CSF for 18 h and then pulsed with TAA derived from S1509a. This resulted in protective immunity against subsequent tumor challenge, providing a model to study the conditions required for sensitization against TAAs by epiderma! APCs. Culture of ECs in GM-CSF was required for induction of significant protective tumor immunity, and Uv irradiation or incubation in TNF-a for 2 h after GMCSF incubation abrogated the immunostimulatory effect of GM-CSF. However, unlike UVR, TNF-a did not significantly inhibit the induction of immunity when ECs were exposed to TNF-a before overnight incubation in GM-CSF, together with GM-CSF, or after pulsing with TAA, and anti-TNF-a antibody treatment did not abrogate the effects of UVR on this system. Furthermore, TNF-a incubation of ECs augmented their ability to elicit delayed-type hypersensitivity (DTH) and also enhanced elicitation of DTH by GM-CSF-cultured ECs,
Medical
skin tumors (reviewed about the mechanisms
regulation
antigen-presenting
C. Tan, and
Germany
and carcinogen-induced tle information exists itiation
Kong
Harvard
Miinster,
significance.
whereas UV-irradiation fashion. Taken together, GM-CSF, TNF-a, and tumor antigen presentation
Hosoi,
of Dermatology, of Miinster,
ultraviolet radiation (UVR) and the cytokines granulocyte-macrophage colony-stimulating factor (GMCSF) and tumor necrosis factor a (TNF-a) on the ability of epidermal cells (ECs) to induce or to elicit immunity against the murine spindle cell tumor S1509a. Naive syngeneic mice were immunized three times at weekly inter-
Key
and
a,
a;
ltF-fl, UVR,
Grabbe,
LC,
accepted
transforming ultraviolet
at the
Seattle,
Volume
factor
radiation.
13th 16,
Street,
Hospital,
Charlestown,
1992.
annual
May
tumor-
growth General
Center, March
presented
Langerhans TAA,
Massachusetts
Research
sorter; colony-
meeting
of the
Soci-
1991.
52,
August
1992
209
strain
[23].
TNF-a
also
prolongs
without hancing
altering their surface LC antigen presentation
suggests
that
TNF-a
presentation
may
when
LC
viability
marker [9].
down-regulate
given
in
vivo
in
vitro,
characteristics Preliminary cutaneous
[24],
but
but
tamed from Cedarlane Laboratories (Hornby, Ontario) and used at 1:30 in phosphate-buffered saline (PBS) containing 5% FCS. Enzymes used during preparation and dissociation
or enevidence antigen
careful
studies
of
the are
effects of these cytokines on the functional abilities of LC still missing. Furthermore, ultraviolet radiation (UVR), besides being one of the most relevant human cutaneous carcinogens in vivo, is a powerful immunomodulatory agent. UVR has been shown to alter LC function directly as well as to induce cytokine
release
creased
by
levels
skin,
and
the
observed
tion
after
cutaneous
of TNF-a
it has
been
elements.
have
been
suggested
that
down-regulation ultraviolet
TNF-a
other
[25,
cytokines including
example,
in
in-
antigen 26].
In
Thus,
cutaneous
APC
function
immunomodulatory
cytokine
microenvironment
vestigated and TNF-a primary
the
of
of UVR ability
secondary
be
addition,
(ECs) IL-6,
the
skin.
and of the of epidermal
antitumor
affected
and
by This
immune
by
the
local
study
cytokines APCs
in-
GM-CSF to induce
responses
in
syn-
recipients.
MATERIALS
AND METHODS
Mice Female (BALB/c x A/J) F1 i2 weeks old were obtained (Bar
Harbor,
(CAF1) from
(H2(/H2a) the Jackson
mice 6 to Laboratory
ME).
Tumors The
Si509a
line,
originally
by
Dr.
methylcholanthrene-induced derived
Mark
I.
Philadelphia. and 5% CO2
It in
from
spindle A/J
Greene,
0.1
mM
essential
was
University
was maintained RPMI i640
and
cell
kindly of
in tissue supplemented
inactivated fetal calf serum Grand Island, NY), 100 U/ml tomycin,
mice,
tumor
provided
Pennsylvania,
culture with
at iO%
37#{176}C heat-
(FCS) (Gibco Laboratories, penicillin, iOO g/ml strepnonessential
amino
mM L-glutamine, 1 mM sodium pyruvate, HEPES buffer (complete medium). Si509a progressively in normal syngeneic recipients been demonstrated to induce a variety of responses in the host [40].
acids,
2
and 0.01 M usually grows [7] and has immunological
Reagents Cytokines used in this study CSF (Genzyme, Cambridge, TNF-a (Genentech, San monoclonal antibody (clone New
clonal and
210
England
Nuclear,
rabbit used
at
antimouse 1:1000.
Journal
Boston,
TNF-a Low-toxicity
of Leukocyte
included MA) and Francisco, NEI-OOi)
natural murine GMrecombinant human CA). Anti-Thy 1.2 was obtained from
MA
and
was
obtained
rabbit
Biology
used
at
1:1000.
from
complement
Volume
Poly-
Genzyme was
52,
Indianapolis, MO).
lysates (tumor fragments, TFs) as sources of antigens were prepared from freeze-thaw lytumor cells as described elsewhere [7]. cells (i07/ml in complete medium) were disfreeze-thaw cycles and centrifuged at 600g for supernatant was collected and spun again at
i
h.
The
soluble
Preparation Protocol
the
cells IL-3,
greatly
UVR
within
effects on the
and
geneic
may
effects
of
Mannheim, St. Louis,
remaining
supernatant
was
used
as
TFs.
for
and IL-8, is well documented [14, 25, 27-29], and increased serum levels of IL-i [30, 31], IL-6 [32], and TNF-a [25] have been shown after UVR. Direct effects of UVR on cutaneous LCs include decreased antigen presentation to T helper 1 but not to T helper 2 cells in vitro, altered morphology and surface characteristics of LCs in vivo and in vitro, and decreased numbers of LCs in vivo after UV exposure
the
for
source
presenta-
by epidermal GM-CSF, IL-i,
dispase (Boehringer and trypsin (Sigma,
Tumor cell soluble tumor sates of Si509a Briefly, Si509a rupted by four 10 mm. The i3000g
UV-irradiated
is responsible
ofcutaneous
irradiation
release of various after UV irradiation,
For
found
of ECs were IN), DNase,
August
ob-
1992
ECs
were
[7].
Briefly,
(Neet,
prepared
skins
a standard of shaved
Laboratories,
and
depleted
carnosus.
1.5 U/ml
Cells and Immunization
using
truncal
Whitehall
removed ulus
of Epidermal
in
New
Ca2/Mg2-free
as
chemically York,
of subcutaneous skins were floated
The
dispase
protocol and
PBS
depilated
NY)
(s.c.) dermis for
fat 30
described mice
were
and pannicside down on mm
at
37#{176}C,
and epidermal sheets were collected and dissociated by incubation in 80 tg/ml DNase in Ca2/Mg2-free PBS for 20 mm at 37#{176}Cunder continuous gentle agitation, filtered through nylon gauze (Nitex, Tetco, Elmsford, NY), and washed. Thy i-bearing cells were deleted by incubation in anti-Thy i.2 monoclonal antibody solution for 30 mm on ice, followed by washing and subsequent incubation in low-toxicity rabbit complement for 35 mm at 37#{176}C.Dead cells were removed by treatment with 0.05% trypsin and 80 g/ml DNase in Ca2/Mg2-free PBS for 10 mm at 37#{176}C.The percentage of I-Ak cells within the EC population ranged from 8.4 to i7.8%. Viability and percentage of I-At cells were assessed by fluorescence-activated cell sorter (FACS) analysis immediately before injection into mice, and differences between groups within experiments were negligible (data not shown). ECs were then incubated in 50 U/ml GM-CSF and/or 100 U/ml TNF-a in complete medium at 37#{176}C.These cytokine concentrations have been reported to have maximal effects on LC viability in vitro [9]. Control cells were placed for the same amount of time in complete medium alone but, to preserve the functional state of fresh LCs, at 4#{176}C.In other experiments fresh ECs were immediately pulsed with TFs without preculture in GM-CSF or medium alone, yielding the
same
results
as
after
overnight
incubation
at
4#{176}C. After
depletion ofdead cells, the cells were washed three times and incubated in a suspension containing TFs from 1 x 10’ Si509a tumor cells or in complete medium alone for 2 h at 37#{176}C.After TF pulsing, the ECs were washed four times to remove soluble tumor antigen. ECs (2-3 x 105) were then injected s.c. into naive recipient mice on the lower back. This immunization was repeated twice at weekly intervals. One week after the last immunization, mice were challenged with 2 x 106 live S1509a fibrosarcoma cells s.c. on the lower lateral abdomen and tumor growth was assessed every 48 h by measurement with a vernier caliper. Earlier studies showed that this immunization protocol generates tumor immunity in immunized mice, leading to immunologic rejection of the tumor over a period of 10-14 days. The specificity of tumor immunity in this system was previously demonstrated by showing that immunization with tumor cell lysates from an unrelated tumor line (UV-5496-i)
does
not
lead
to immunity
against
S1509a
[7].
Elicitation of S1509a Tumor Immunity of Delayed-Type Hypersensitivity Mice 0.5-1.0
were immunized x 106 dead
thawing) s.c. tive immunity
at
against S1509a
and Measurement by by
three injections of repetitive freeze-
intervals. Generation of protecwas confirmed by rejection of a
subsequent tumor challenge and induction of delayed-type hypersensitivity (DTH) against this tumor (data not shown). ECs from naive donor mice were generated as described above. To delete Thy i.2 dendritic epidermal T cells and some keratinocytes, ECs were incubated in anti-Thy 1.2 monoclonal antibody at 1:1000. To further enrich for epidermal LCs, normal human AB serum at 1:10 was added, because antibodies in human serum have been found to bind to surface glycoproteins on mouse keratinocytes [4i]. Incubation was performed for 30 mm on ice, and the cells were washed twice and incubated in low-toxicity rabbit complement at i:30 for 35 mm at 37#{176}C.Dead cells were deleted as described above. This procedure yielded approximately a 10-fold enrichment of I-Ak cells within the EC preparation (as assessed by FACS analysis), ranging from 13.3 to 26.7% after complement-mediated lysis and depletion of dead cells. ECs were then pulsed with Si509a TFs for 3 h and washed extensively, and 5 x i0 ECs were injected into one hind footpad of mice previously immunized against Si509a. Some groups of ECs were cytokine treated for 3 h or UV irradiated
1000 R
800
I
A: GM-CSF
-.---
B: TNF-X
-0--
C: No Cytokine
-0--
D: GM-CSF
iT
->
after
TF
pulsing.
Specific
( 4#{176}C ) -> No
->
,0
iT
UV Irradiation After
incubation,
dead
of TNF-a
cells
as
distance
above
was
were
thick-
Activity
deleted
by DNase-
ECs
and
of irradiation.
Following
cubated in complete medium for neutralizing antibodies, ECs were of anti-TNF-a antibody at 1:1000 radiation, and all further incubations presence
swelling
the footpad side.
were washed three UV irradiation, ECs were resuspended in 3 ml of placed into 60-mm2 petri dishes (Falcon, Lincoln and exposed to an FS4O sunlamp (Westinghouse, PA) emitting approximately 5.1 W/cm2 of UVB
treatment
times. For clear PBS, Park, NJ), Pittsburgh, at the
and Neutralization
cytokine
trypsin
footpad
between uninjected
of anti-TNF-a
cells
were
in-
antibody.
Data Generation
and Statistical
Experiments
performed
were
UVR,
1 h. In experiments using incubated in the presence immediately after UV irwere performed in the
Evaluation
at least
three
times.
The
tumor
volume was calculated as the product of the maximal tumor diameter in three perpendicular directions, measured with a vernier caliper. This method has previously been confirmed to correlate well with the tumor weight [7]. To avoid unnecessary pain to the experimental animals, mice were sacrificed when the tumor volume exceeded 1000 mm3. To evaluate statistical differences between the mean tumor volume in the various experimental groups, a two-way analysis of variance based on the slopes of the regression curves in each group was performed. The Sidak multiple comparison procedure was used for pairwise comparison of all groups, using a P value of less than .05 to determine significance, since this multiple comparison procedure does not specify P values for each pairwise comparison [42]. DTH results were analyzed using Student’s t-test for independent samples.
iT
->
or
measured as the mean difference ness of the injected versus the
Si509a (killed
5- to 7-day in these mice
before
iT
n=5/group
RESULTS 600
EC-Mediated by Incubation
z 200
0
0
5 DAYS
1. Modulation
munity
by 50
medium
alone
2
x l0
of three cells
but of
in mice
C, D, P
iT
B: GM-CSF
->
No
800
.
A: GM-CSF
S
C TNF-a
II
D:
iT -D--
CGM-CSF+TNF-a->iT
B: GM-CSF
(.5
8 8
n=5/group
E
->
iT
->
No
iT
GM-CSF
->
GM-CSF
TNF-
->
iT
->
cx
iT
->
‘U
‘U
600
n=5/group 0
0
400 0 I-
z .‘
400
:
‘U
200 200
0
5
10
DAYS
C
1000
AFTER
15
TUMOR
.
A:
->
iT
U
B: GM-CSF
->
iT
0
->
No
-D--
GM-GSF
GM-cSF
20
0
5
CHALLENGE
DAYS
10
AFTER
15
TUMOR
CHALLENGE
TNF-a
->
iT
800. (‘5
n=5/group
E 8 ‘U
Fig. 2. Differential effect of TNF-a on EC-mediated immunity against Sl509a. (a) GM-CSF augments induction of immunity against Sl509a even in the presence of TNF-a. The graph shows the mean tumor volumes in mice immunized with ECs cultured in 50 U/mI GM-CSF for 18 h, followed by a 2-h pulse with TFs (A, positive control) or culture medium alone (B, negative control), or with ECs that were coincubated in 50 U/ml GM-CSF
600 0
400
and
100
U/ml
P < .05;
TNF-a,
A vs. C,
abrogates
the
ability
munity.
ECs
were
followed
by
incubation
followed
N.S.
(b)
by
a 2-h
Exposure
pulse
ofGM-CSF-cultured
cultured
incubated
in TFs 100
(A) U/ml
presence or
medium
and
D were
TNF-a
tion
in 50 U/mI GM-CSF, followed by a 2-h A vs. C, B vs. D, N.S. (c) TNF-a
P < 0.05; tured
0
5 DAYS
212
Journal
of Leukocyte
10 AFTER
TUMOR
Biology
15
20
CHALLENGE
Volume
52,
August
1992
ECs
when
applied
after
antigen
exposure.
TFs
after
ECs
in the
with
with
to TNF-a
(C).
culture
A,
C
to induce
S1509a
tumor
of 50 U/mI
GM-CSF
for
alone for
(B)
for
2 h before
pulse with incubation ECs
vs.
B,
in GM-CSF
2 h. or
after
im-
18 h,
Groups
C
incuba-
TFs. A, C vs. B, D, does not affect culwere
incubated
in
50
U/mI GM-C5F for 18 h, pulsed with TFs for 2 h, and then treated with 100 U/mI TNF-a for 2 h (A). Controls were incubated in 50 U/mI GM-CSF for 18 h, followed by incubation in TFs (B) or medium alone (C). Graphs show the mean tumor volumes in mice immunized with these differentially treated ECs. A, B vs. C, P < .05; A vs. B, N.S.
cubation in 50 U/ml GM-CSF for 18 h did not affect the observed ability of GM-CSF to augment the capability of ECs to induce tumor immunity in vivo. However, incubation of
at doses
ECs in iOO U/ml pletely abrogated
were still recipient
5i509a ligated when
TNF-a after exposure the ability of TF-pulsed
tumor immunity in vivo whether TNF-a affects administered to ECs after
2c shows, no significant ECs were incubated We conclude that
(Fig. tumor antigen
to GM-CSF ECs to
corninduce
2b). Finally, we invesantigen presentation exposure. As Figure
effect of TNF-a in 100 U/ml TNF-a TNF-a is capable
below
800
on the tumor immunization.
was observed when after TF pulsing. of down-regulating
J/m2.
antigen, ECs
To control
TFs pulsed
able to mediate mice, and the
for
immunity short-term was not shown).
We antigen
that UVR, EC-mediated
tumor
conclude inhibits
immunity
in
effects
against viability
sessed by FACS analysis) UV treatment (data not therefore exposure,
direct
of UVR
were UV irradiated and with these UV-irradiated
used
for TFs
S1509a in normal of I-Ak ECs (as-
significantly
affected
by
applied before or after induction of Si509a
a dose-dependent
manner.
the GM-CSF-induced augmentation of EC tumor antigen presentation when cells are exposed to TNF-a after GMCSF incubation but before TF pulsing. These results also demonstrate that the effect of GM-CSF on the ability of ECs to mediate tumor immunity is reversible.
TNF-a Is Not the Principal Mediator for UVR-lnduced Inhibition of EC Tumor Antigen Presentation in Vitro
UV Irradiation of TF-Pulsed ECs Inhibits Their Ability to Generate Tumor Immunity in a Dose-Dependent Manner
production regulatory
Because posure, immunity,
tumor clonal radiated
Because UVR adversely affects EC antigen presentation and induces TNF-a secretion by epidermal keratinocytes [26], we assessed the effect of UVR on the induction of tumor immunity by TF-pulsed ECs. It was found that FS4O sunlamp
both UVR down-regulated we were
and
TNF-a, given before antigen cxEC-mediated generation of tumor interested in whether UVR-induced
of TNF-a was responsible effects of UVR on EC-mediated immunity. To test this possibility, rabbit antimouse TNF-a antibody ECs immediately following UVR.
b U
800
-0A
I
A: GM-SF
->
iT
B: GM-CSF
->
No
Q GM-CSF
->
200J/m2->
Th GM-cSF
->
400J/m2
E: GM-SF
->
A: GM-SF
->
iT
B: GM-CSF
->
No
-0-
iT
.
iT ->
iT
(.5
neutralizing was added All further
polyto irincu-
iT
QGM-cSF->iT
->
200J/m2
ThGM-CSF->iT
->
400J/m2
EGM-SF->iT
->
800J/m2
8 8
iT
800J/m2->
the downgeneration of
bations after UVR exposure in these experiments were performed in the presence of anti-TNF-a antibody. The effect of anti-TNF-a on the inhibition of EC-mediated tumor immunity by two different doses of UVR is shown in Figure 4a (200 J/m2) and 4b (400 J/m2). Although a trend toward smaller tumor volumes in the groups that received anti-TNF-a was evident in two of three experiments performed, the inhibition of tumor growth was not statistically significant when compared to nontreated irradiated ECs.
irradiation of GM-CSF-cultured ECs prior to exposure to TFs inhibited the induction of immunity against Si509a by ECs in a dose-dependent manner (Fig. 3a). In contrast to TNF-a incubation, UV irradiation of ECs after exposure to TFs also affected their ability to present Si509a tumor antigen and to induce in vivo protective immunity (Fig. 3b), although only partial abrogation of immunity was observed
1000
for
‘U n=5/group
n=5/group
600
400
200 100
0 0 DAYS
Fig.
3. UVR
irradiated Controls
inhibits
with were
with
the
various not
in mice immunized were immunized munized
10
5
ability doses
UV
AFfER
irradiated
of
TUMOR
to induce
UVR,
rested
and
pulsed
differentially
treated
20
0
5 DAYS
CHALLENGE
of ECs
with these differentially with ECs treated as above,
these
15
in with
immunity culture medium
against medium
Sl509a for
containing
1 h, TFs
in a dose-dependent and (A)
incubated or
medium
manner. in
medium alone
TUMOR
for
2 h.
TFs The
as graph
treated ECs. A vs. B, P < .05; D, E vs. A, P < .05; C vs. A, B, N.S.; but ECs were pulsed with TF before UV irradiation. The graphs show ECs. A vs. B, P < .05; C, D, E vs. A, B, N.S.
Grabbe
et al.
Cytokines
and
UV
modify
tumor
D,
the
a source shows
(18 h, 50 U/ml) ECs were of TAA for 2 h (C-E). the
E vs.
B,
mean
tumor
antigen
15
CHALLENGE
(a) GM-CSF-cultured containing
(B)
10
AlTER
N.S.
mean (b)
tumor Groups
volumes
presentation
volume of
in mice
mice
im-
213
b 400
.
B: No iT
-A-
A
300
A: iT
A:iT
-0#{149}--
Q200J/m2
->iT
D’.200J/m2
->NoiT
E 200J/m2
antl-i”NF-a
->
E: 4002
0
5 DAYS
Fig. CSF
4. Anti-TNF-a for 18 h, and
TFs
for
2 h. The
(A)
10
AFTER
TUMOR
0 DAYS
control
or not
groups
pulsed
(A
with
medium
and
TF
(D).
B) show
(B). In
the
Groups
group
(E),
mean
(C-E)
tumor
were
volumes
treated
anti-TNF-a
antibody
over
time
identically, (1:1000)
The the
quantity of antibody added was sufficient to neutralize production of approximately 500 U of TNF-a by each 106 ECs, well above the reported levels of TNFa released by keratinocytes after UVR exposure [25]. No significant amounts of TNF-a were found in supernatants of UVirradiated and antibody-treated ECs, using a standard TNFa-sensitive cell line (WEHI 164.1) [43] (data not shown). We therefore conclude that UVR and TNF-a inhibit ECmediated generation of tumor immunity via at least partially different mechanisms.
Incubation of ECs in TNF-a Enhances S1509a Tumor Immunity by TF-Pulsed Tumor-Immune Mice Because TNFa tion of Si509a effect by
sayed
and tumor
ofthese fresh
immune
or
mice. by
tion of presence
GM-CSF immunity
the Elicitation ECs in
profoundly by ECs,
affected we also
the inducinvestigated
cytokines on the elicitation oftumor GM-CSF-cultured ECs in primed, The ability of ECs to present TAA
measuring
the
TF-pulsed or absence
DTH
ECs that of cytokines
response
had
elicited
been of interest
of
by
incubated either
immutumorwas asthe injecin before
the or
after TF-pulsing. Results obtained using freshly prepared, LC-enriched ECs as APCs are shown in Figure 5a. In contrast to the findings obtained for the induction of immunity (see above), incubation of fresh ECs in TNF-a (100 U/ml, 3 h) enhanced S1509a-specific DTH. The effect of GM-CSF and TNF-a was independent ofthe order ofexposure of ECs to the cytokine or TFs because no significant differences in
214
iT
->
Journal
of Leukocyte
Biology
Volume
52,
August
1992
10
AFTER
TUMOR
CHALLENGE
of epidermal cell antigen presentation. ECs were incubated in 50 U/ml UVR, followed by incubation in complete medium for 1 h and pulsing in mice
but was
added
UV
immunized
with
irradiated, to the
ECs
rested
culture
that
were
in medium
medium
out all following incubations including TF pulsing. The graphs show the mean tumor volumes in mice (a) A vs. B, P < .05; B vs. C vs. D vs. E, N.S. (b) A vs. B, P < .05; B vs. C vs. D vs. E, N.S.
nity
anti-TNF-a
5
CHALLENGE
does not significantly affect the UVR-induced inhibition some groups were exposed to (a) 200 J/m2 or (b) 400 J/m
or complete
the
>
I
100
(C)
->iT ->NoiT
200
200
TFs
Q400J/m2 D’.400J/m2
n=5/group
n=5/gmup
TF
-A(‘5
8 8
with
B: No iT
A
iT
->
-0--
not
alone
immediately
immunized
irradiated
for after
with
these
but
I h, and
were
GMwith pulsed
incubated
irradiation
and
differentially
with through-
treated
ECs.
the amount of inhibition or stimulation were observed when TNF-a was added before or after antigen exposure (Fig. 5a). Similar results were obtained using cultured instead of fresh ECs (ECs were enriched for I-Ak cells as described above and cultured in 50 U/ml GM-CSF for i8 h). As demonstrated in Figure 5b, incubation of cultured ECs in 100 U/ml TNF-a also augmented their ability to present S1509a TAA for the elicitation of tumor-specific DTH in three of four separate experiments, whereas it had no effect in one experiment. No specific footpad swelling was induced by injection of equal numbers of cytokine-incubated but not TF-pulsed ECs, indicating that carryover of cytokine into recipient mice did not interfere with the DTH measurements. Likewise, injection of cytokine-treated ECs into nonimmune mice did not induce significant footpad swelling (data not shown). We therefore conclude that the elicitation of immunity by TF-pulsed ECs is enhanced by incubation of ECs in TNF-a before or after exposure to TFs.
UVR Blocks ECs We were also present tumor vivo is affected UV TFs ously
shown munity
Elicitation
immunized against
by TF-Pulsed
interested in whether the ability antigen to primed, tumor-immune by UVR. To test this hypothesis,
irradiated either and injected into in Figure
of Tumor Immunity
before or syngeneic
against
after they recipients
S1509a
6a,
UVR
Si509a
in
suppressed a dose-dependent
as
were that
of
ECs to mice in ECs were
pulsed were
described
the
with previ-
above.
elicitation fashion.
As
of imECs
ir-
a
b
AuguseMetion
%
% Augmentation GM-CSF -> TV
#{149}1
GM-CSF
No TV
GM-CSF
TF->TNF-a
#{149}U#{149}t-i
a->
TNF-
+53.1
-I
:_
->
TNF-a
1
TV
BI
No TV
->
->TF
H
NoTF
TNF-a->
H Non-
NoTF
1’
__________
a
. 20
SPECIFIC
5. Effects
of 1 x
of TNF-a
dead
106
(5
x
l0
cells
in the presence
EC/mouse).
The
of
graph
100
was
U/ml
shows
ECs
proved
TNF-a
the
for
DTH
______ ________ _________
S
TF
->
#{149}
.
#{149}
50 SPECIFIC
to elicit by their
a tumor-specific ability
3 h before
response
n-S/group
0
1-’-
1102
of TF-pulsed
immunity
#{149}
40
SWELLING
ability
and
I
‘
30
FOOTPAD
on the
Sl509a
were incubated
#{149}1
o
0
Fig.
immunized mice
->TNF-a
or
elicited
by
to reject after these
TF
FOOTPAD
DTH
response.
Mice
were
a tumor
challenge
with
2 x
pulsing,
washed,
differentially
and
treated
injected
ECs
immunized live
106
into
as the
SWELLING
one
mean
against S1509a
mm)
110 S1509a
cells.
hind
footpad
differences
in
(a)
by
ECs
s.c.
injections
enriched
for
of tumor-immune footpad
LC mice
thickness
between
alone (groups A, B, E). ECs were (3 x l05/mouse). Footpad swelling
P < .001; A vs. C, P < .001; A vs. D: P < .001; B vs. E: N.S. (b) ECs were enriched for LCs GM-CSF for 18 h. Nonadherent cultured ECs were incubated in 100 U/ml TNF-a for 3 h (groups C, D) or in medium then pulsed with TFs (A, C, E) or medium alone (B, D) for 3 h, washed extensively, and injected into one hind footpad was measured as described above. A vs. B, P < .001; A vs. C, P = .003; B vs. D, E: N.S. Numbers indicate % differences
in specific
background.
the
injected
as above
and
and
the
uninjected
cultured
DTH
foot
in 50 U/mI
response
minus
after
24
h.
A
vs.
B,
b
% Suppiession
200j/m2
TF
D
NoTF
->17
c__
50J/m2
->17
._
->
200J/m2
->17
E
TV
i
57-9
200J/m2
->17
73.9
iooyin2
..>TF
Ii
TF
92.0
->
6L2
48.9
F
32.4
=
G
in2
200
-I
D
TV
50J1m2->
>100
-I
400 JIm2 ->17
0
NoTF
2&4
H
H
A
No TF
->
100J/m2->NoTF
25J/n?
100 JIm2
% Suppi.ion
75.6
n-S/group S00J/m2
g:-.i
->17
TF->
>100
0
10 SPECIFIC
20 FOOTPAD
30
SWELLING
40
TF
->
-
J/m2
100
[_
JIm2
50
1
11112
58.0
_______
25.6
TF
No TF NonImmunized Mice
->
M
TV
na’S/group
I .
,.I.I.I.
0
I
10
20
30
SPECIFIC
FOOTPAD
50
40
SWELLING
110
-2
mm)
Fig. 6. Effects of UVR on the ability of TF-pulsed ECs to elicit a tumor-specific DTH response. Tumor-immune mice were prepared as described. (a) LC-enriched ECs were irradiated with varying doses of UVR and incubated in complete medium for 1 h before TF pulsing. A vs. B, P = .005; A vs. C, N.S.; A vs. D, P = .048; A vs. E, P = .01; A vs. F-H, P < .01. (b) Effect of UV irradiation of ECs before or after exposure to TFs. The graphs show the DTH response elicited by the injection of 5 x 10 ECs/mouse into hind footpads of tumor-immune mice, measured as the mean differences in footpad thickness between the B, C, M, N.S. Numbers
injected indicate
and
the %
uninjected differences
foot in
after
specific
24 h. K vs. A, B, C, DTH
response
Grabbe
et al.
minus
D, E, G, H,
L, M,
P
