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Immunology |
Malaghan Institute of Medical Research, Wellington School of Medicine, Wellington South, New Zealand [P. R., I. F. H., S. P. H., J. M. R., F. R.], and Department of Immunology, Institute of Medical Microbiology and Hygiene, University of Freiburg, Freiburg, Germany [H. P.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The potential advantages of using unfractionated tumor peptides as vaccines are counterbalanced by a number of drawbacks that this nonspecific strategy can encounter in its practical application. The level of expression of the tumor peptide on tumor cells is probably the most important issue. Because tumor cells are used first as a source of antigen and later become targets of the antitumor immune response, limited presentation of tumor antigen becomes critical at two separate stages. Other factors that can limit the efficacy of this approach include the immunogenicity of the tumor peptides and the presence of specific T-cell precursors (3) . Because tumor-associated peptides are essentially self-antigens, they are possibly weakly immunogenic or not immunogenic at all. Immunodominance of some antigens over others can lead to failure to elicit some T-cell responses (4) , although the use of BMDCs3 as antigen carriers possibly abrogates dominance features among antigens (5) . Additionally, other issues that have to be considered are more quantitative and concern the total amount of peptide that can be isolated from tumor cells. Efficient T-cell recognition seems to be critically dependent on adequate tumor antigen concentration (6 , 7) . However, a frequent feature in tumor biology is down-regulation of MHC class I molecules (8 , 9) , which leads to a general scarcity of MHC class I-associated peptides and altered recognition of tumor cells by CTLs (10) .
In consideration of these restrictions, it is reasonable to hypothesize that the efficacy of using unfractionated tumor peptides as immunogens can be easily compromised by the low concentration of effective tumor peptides on tumor cells (11) .
Our aim in this study was to investigate whether the relative abundance of tumor-specific peptides expressed on tumor cells was a limiting factor in the induction of antitumor immune responses. We have adopted an experimental system where different tumor cell lines were transfected to express one MHC class I-associated peptide, LCMV33–41 (12 , 13) . The same synthetic LCMV33–41 peptide can be used together with DCs to induce antitumor immune responses (13) . In this context, we have explored the efficacy of using unfractionated peptides isolated from these tumor cell lines expressing different levels of LCMV33–41.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice and Cell Lines.
C57BL/6J mice (Jackson Laboratories, Bar Harbor, Maine) and "318"
mice (14)
, transgenic for a T-cell receptor specific for
Db+ LCMV33–41, were
maintained in the Animal Facility of the Wellington School of Medicine
by brother x sister mating. The Lewis lung carcinoma
LLTC, the murine thymoma EL4, and the murine melanoma B16.F10 were
maintained by weekly subculturing in CM. LL-LCMV and EL-LCMV are
transfectants of LLTC and EL4, respectively, generated by expressing a
minigene encoding the LCMV glycoprotein amino acids 33–41 under the
control of a cytomegalovirus promoter as described
(13)
. B16.F10GP33 was derived from
parental B16.F10 by transfection of the GP33 LCMV minigene
(12)
. The transfected cell lines LL-LCMV, EL-LCMV, and
B16.F10GP33 were maintained in CM containing 0.5,
0.4, or 0.2 mg/ml G418, respectively (Life Technologies).
Growth of BM-derived DC.
DCs were generated in culture from bone marrow precursors in the
presence of IL-4 and granulocyte/macrophage-colony-stimulating factor
as described (15)
. Cultures were fed fresh medium and
lymphokines every 2–3 days and incubated at 37°C for 6–7 days.
Flow Cytometry.
Fluorescence-activated cell sorter analysis was performed as described
previously (15)
. The
anti-H-2Db-biotin monoclonal antibody was
purchased from PharMingen (San Diego, CA).
Tumor Peptide Preparation.
Peptides were isolated from tumor cell lines as described
(16)
. Briefly, up to 108 cells were
washed three times in PBS (Life Technologies, Inc.) and incubated with
10 ml of acid buffer (0.131 M citrate, 0.066 M
Na2HPO4, pH 3) for 1 min.
Adherent cell lines (LL-LCMV and B16.F10GP33)
were trypsinized and washed prior to the elution. Cell suspensions were
then pelleted, and the supernatants were clarified by centrifugation at
2000 rpm for 5 min. The peptides in citrate-phosphate buffer were
concentrated on activated SepPack C18 cartridges
(Waters, Milford, MA). The cartridges were prewashed with 5 ml of
acetonitrile and then with 5 ml of double-distilled
H2O. Acid extracts were then loaded and allowed
to gravitate through the cartridges. After washing with 3 ml of
double-distilled H2O, the bound material was
eluted in 5 ml of 60% acetonitrile. This material was subsequently
lyophilized in a Sped Vac evaporator-concentrator (Savant). Lyophilized
peptides were stored at -70°C until further use.
LCMV33–41-specific Proliferation Assays.
The proliferative response of LCMV33–41-specific
T cells to DCs pulsed with tumor peptide extracts or with synthetic
LCMV33–41 peptide was determined by incubating
2 x 105 spleen cells from
"318" mice with serially diluted DCs that had been pulsed with
antigen for 3 h at 37°C. Proliferative responses to tumor cell
lines were determined by incubating 5 x 105 "318" spleen cells with serially diluted
mitomycin C-treated tumor cells in the presence of 10 units/ml of IL-2
as described (13)
.
Cytotoxicity Assay.
Spleen cells from "318" mice were cultured for 5 days in 24-well
plates (Falcon) in CM in the presence of 0.01 µM
LCMV33–41 peptide and 20 units/ml of IL-2 and
fed every second day with CM and IL-2. At day 5, viable cells were
washed, counted, and assayed for CTL activity by the JAM test
(17)
as described (13)
. In some experiments,
a 51Cr-release assay was carried out as described
(12)
.
In Vivo Tumor Protection.
DCs (106 cells/ml) cultured for 6 days were
incubated overnight at 37°C in CM in the presence of 20 ng/ml IL-4
and granulocyte/macrophage-colony stimulating factor and either 10
µM LCMV33–41 peptide or tumor
peptide extracts. DCs were washed three times, resuspended at
106 cells/ml in Iscove’s modified Dulbecco’s
medium, and used for immunization. Groups of C57BL/6J mice
(n = 5) were immunized s.c. into the right
flank with 105 peptide-loaded or control DCs on
days 0 and 7. After 7 days from the last immunization, all animals were
challenged with 2 x 105 LL-LCMV
or B16.F10GP33 tumor cells, injected s.c. in the
opposite flank. Mice were monitored every 3–4 days, and mean tumor
size for each group was calculated as the mean of the products of
bisecting tumor diameters as described. Measurements were terminated
when the tumor size of one mouse in each group reached 200
mm2 or whenever animals were visibly unwell. All
experiments were approved by the Wellington School of Medicine Animal
Ethics Committee and performed in accordance with the University of
Otago guidelines.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows that expression of H-2Db on EL-LCMV and
LL-LCMV cells is similar, whereas B16.F10GP33
express very low levels of H-2Db. This is
consistent with what was reported previously for the parental B16.F10
cell line (18)
. An
LCMV33–41-specific proliferation assay (Fig. 2A
) showed that EL-LCMV and B16.F10GP33
cells stimulated similar proliferation of
LCMV33–41-specific T cells, whereas LL-LCMV
cells had weaker stimulatory capacity. These differences in stimulatory
capacity were most likely attributable to differences in presentation
of LCMV33–41, because the assay was carried out
in the presence of IL-2 to minimize the effect of costimulatory
molecules that might be differentially expressed by the tumor cell
lines. A similar conclusion was also suggested by the results of the
cytotoxicity test shown in Fig. 2B
.
B16.F10GP33 cells were lysed by
LCMV33–41-specific CTLs much more efficiently
than LL-LCMV tumor cells, suggesting a higher presentation of specific
antigen on B16.F10GP33 cells. EL-LCMV cells were
not tested in this assay.
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In Vitro LCMV33–41-specific Bioassays
Fail to Detect Eluted LCMV33–41 Peptide.
We wished to establish whether the acid elution approach was effective
in recovering MHC class I-associated peptide, even when expression of
class I is low (B16.F10GP33) or when the amount
of tumor peptide presented on the tumor cell is relatively small
(LL-LCMV). The EL-LCMV cell line provided an optimal model for this
type of antigen isolation, having both high levels of
H-2Db and a good ability to stimulate
LCMV33–41 peptide-specific responses, whereas
the B16.F10GP33 and the LL-LCMV cell lines would
represent more physiological systems.
Peptide mixtures eluted from the
LCMV33–41transfected cell lines were tested for
their ability to stimulate proliferation and cytotoxic activity of
LCMV33–41-specific T cells in vitro.
These assays were chosen as the most sensitive, because T cells can
respond even to very few antigen molecules presented on a cell
(19)
. However, in both
LCMV33–41-specific proliferation and
cytotoxicity assays (Fig. 3
) the peptide preparations obtained from
B16.F10GP33, EL-LCMV, and LL-LCMV cells were
unable to elicit a response. This is despite the presence of
considerable amounts of MHC class I-derived peptides in the
preparations used, as shown by their ability to induce the renaturation
of surface MHC class I on the mutant cell line RMA-S (data not shown).
This observation raised the question of whether
LCMV33–41-specific bioassays are sensitive
enough to detect the small amounts of LCMV33–41
peptide that can be theoretically isolated from the transfected
cell lines.
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1000
different peptides can be presented by class I molecules on a given
cell (20)
. The resulting peptide mixture was then assayed
in a JAM test for its ability to sensitize target cells to killing by
LCMV33–41-specific effect cells, but no
LCMV33–41 specific killing could be demonstrated
(data not shown). The same peptide mixture was also tested in an
LCMV33–41-specific proliferation assay using
BMDCs as stimulator cells, but again no
LCMV33–41-specific proliferation was detected
(data not shown). We conclude that our in vitro tests are
not sensitive enough to detect the very small amount of
LCMV33–41 peptide that could be eluted from the
transfected tumor cell lines.
Irrelevant MHC Class I Binding Peptides Dilute or Inhibit the
T-Cell Stimulatory Activity of LCMV33–41 Peptide in
Vitro.
We then addressed the question of whether other MHC class I-binding
peptides coisolated in the acid elution process could affect the
stimulatory ability of LCMV33–41. Decreasing
amounts of LCMV33–41 peptide were mixed with
irrelevant peptides isolated from the nontransfected EL4 cell line and
tested in a LCMV33–41-specific proliferation
assay (Fig. 4
). At high concentrations of LCMV33–41 peptide,
the presence of irrelevant peptides did not affect the response to
synthetic peptide, whereas at limiting concentrations of
LCMV33–41 peptide, the proliferative response
was inhibited strongly. Thus, the excess of irrelevant peptides present
in the mixtures isolated from tumor cells can exert an inhibitory
effect on poorly represented tumor epitopes in vitro.
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In the case of LL-LCMV, the most effective protection was observed
after immunization with relatively high amounts of
LCMV33–41 synthetic peptide on BMDCs (10
µM, Fig. 5A
and 1
µM, Fig. 5D
).
Protective tumor immunity was also observed after immunization with
BMDC- and LL-LCMV-derived peptides (Fig. 5 and B
). The effect was reproducible and significant over several
experiments (Fig. 5C
) but did not induce complete protection
because all treated mice eventually developed tumors (Fig. 5B
). Peptides derived from the two unrelated tumor cell
lines EL-LCMV and B16.F10GP33, which presumably
only share the model LCMV33–41 tumor peptide
with the LL-LCMV cell line, were less effective at inducing
anti-LL-LCMV immune responses. Peptides from EL-LCMV were essentially
inactive (Fig. 5 and B
). Peptides from
B16.F10GP33 had a small effect in delaying the
onset of LL-LCMV tumor growth (Fig. 5 and E
).
The effect appeared to be attributable to the expression of
LCMV33–41 because peptides from the
nontransfected B16.F10 line were completely ineffective.
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). Again, peptides from the EL-LCMV tumor cell line were completely
ineffective.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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but at the same time are potent stimulators of
LCMV33–41-specific, TCR transgenic T cells (Fig. 2A
) and are efficiently lysed by specific CTLs (Fig. 2B
). These data suggest that the
LCMV33–41 peptide probably occupies a good
proportion of the available MHC class I molecules on these cells. In
contrast, the EL-LCMV and LL-LCMV cells express much higher levels of
MHC class I than B16.F10GP33 cells (Fig. 1
), but
the relative density of LCMV33–41 on their
surface is probably much lower, because EL-LCMV and LL-LCMV have
respectively similar or weaker abilities to stimulate
LCMV33–41-specific T cells as compared with
B16.F10GP33 (Fig. 2A
). Cytotoxicity
data showing that the LL-LCMV cell line is lysed by
LCMV33–41-specific CTLs much less effectively
than the B16.F10GP33 cell line (Fig. 2B
) also supports this.
Regardless of the different expression of the
LCMV33–41 model tumor antigen on these tumor
cell lines, we were unable to demonstrate the presence of
LCMV33–41 in the material eluted from each of
the transfected tumor cell lines by using either an antigen-specific
in vitro T-cell proliferation assay or an in
vitro cytotoxicity assay (Fig. 3
). Assays using T-cell responses
as readouts would be expected to be especially sensitive, because T
cells require only a few antigen molecules/cell to express functional
activity (19)
. However, no activity could be demonstrated
in those assays. Biochemical analyses such as high-performance liquid
chromatography and mass spectrometry also failed to reveal the presence
of the LCMV33–41 peptide in mixtures from the
EL-LCMV tumor cell line. The inability to demonstrate the presence of
LCMV33–41 contrasts with results reported by
another group, where unfractionated peptides derived from
OVA-expressing tumor cells were as effective as the purified OVA
peptide at inducing primary CTL responses in vitro
(1)
. This difference may imply that our tumor models more
closely simulate naturally occurring tumors, where MHC class I
expression is often limited and expression of tumor antigens can be
very low (21)
.
One might assume that the problem of low representation of tumor
peptide on the tumor cell could be resolved by using larger numbers of
cells as a source of antigen. However, this is not always possible when
dealing with ex vivo tumor tissue that may only be available
in limited amounts. Furthermore, irrelevant peptides in the peptide
mixture may inhibit or dilute the effect of the tumor-specific
peptides. Results from in vitro experiments suggest that
recognition of synthetic LCMV33–41 peptide was
inhibited by the presence of other peptides when the concentration of
the relevant peptide was limited (Fig. 4
). A similar process could be
operating in vivo, although we could not test this
possibility directly, because the concentration of
LCMV33–41 at which inhibition by irrelevant
peptide has been observed does not induce tumor protection (data not
shown). The mechanisms of inhibition may be multiple and may be
accounted for by both competition for binding to MHC class I molecules
and inhibition of TCR recognition and/or signaling. Using DCs as
antigen carriers possibly introduces a further limitation, because the
antigen uptake by these cells may be saturable and possibly selective
in the uptake of subsets of antigens. On the other hand, the use of
BMDCs as carriers possibly abrogates dominance features among antigens
(5)
, and therefore antigens that were subdominant when
expressed on tumor cells may become dominant epitopes when presented by
DCs.
Our in vivo tumor protection results suggest that the
efficacy of vaccination using unfractionated tumor peptides is directly
dependent on the relative density of tumor-specific antigen presented
on tumor cells. Good tumor protection was observed when mice were
immunized with the synthetic tumor antigen
LCMV33–41, and some degree of antitumor immune
response was also achieved using peptides obtained from the same cell
line used to challenge mice (Figs. 5
6
). Therefore, an effective
vaccine seems to require either a relatively high amount of a single
purified tumor antigen or a mixture of multiple peptide epitopes.
Interestingly, vaccination with DCs pulsed with peptides isolated from
the B16.F10GP33 cell line delayed the onset of
LL-LCMV tumors, whereas vaccination with EL-LCMV peptides did not have
any effect on tumor growth (Fig. 5
). The protective effect was
attributable to LCMV33–41 in the peptide mixture
and not to other unrelated peptides, because peptide preparations from
the nontransfected B16.F10 cell line were completely ineffective.
B16.F10GP33 and EL-LCMV appear to present
approximately similar levels of LCMV33–41,
because they are equally able to induce the proliferation of specific T
cells in vitro, but their expression of MHC class I
molecules differs significantly. The most likely reason for the
different effectiveness of the two peptide preparations may be the
relative abundance of the LCMV33–41 peptide,
which is likely to be proportionally less represented in the mixtures
from EL-LCMV cells because the expression of
H-2Db is higher on this cell line. Thus, it is
not only the absolute amount of antigen presented on the tumor cell but
also its relative concentration that determines the final effect of
vaccination.
The amount of antigen presented on the tumor cell was also critical in
determining the efficacy of the immune response at protecting against
tumor challenge, as reflected by the amount of
LCMV33–41 peptide needed to pulse DCs to achieve
tumor protection. An optimal antitumor immune response against the
LL-LCMV tumor, which presents low levels of the transfected tumor
antigen LCMV33–41, required DCs to be pulsed
with at least 10 µM synthetic peptide (Fig. 5
). On the
other hand, vaccination with DCs pulsed with 100 nM
synthetic peptide conferred maximum protection against tumor challenge
with the B16.F10GP33 melanoma, which presents a
much higher density of the LCMV33–41 peptide
(Fig. 6
). Protection from challenge with the two tumor cell lines
involves similar effector cell
mechanisms,4
suggesting that the different peptide concentrations do not result in
the generation of qualitatively different effector immune responses.
Rather, this difference may be attributable to the necessity to prime a
more vigorous T-cell response, if the amount of specific antigen on the
target tumor cell is limited.
We conclude from these studies that MHC class I-associated tumor peptides can be obtained directly from tumor cells for use in tumor immunotherapy. However, the relative proportion of tumor peptides presented on the tumor cell is an especially critical factor, because tumor cells are first the source of antigen and later the targets of the in vivo immune response.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by research grants from
the University of Otago, the Health Research Council of New Zealand,
and the Wellington Division of the Cancer Society. 
2 To whom requests for reprints should be
addressed, at Malaghan Institute of Medical Research. P. O. Box 7060,
Wellington, New Zealand. Phone: 64-4-385-5096; Fax: 64-4-389-5095;
E-mail: fronchese{at}malaghan.org.nz
3 The abbreviations used are: BMDC, bone
marrow-derived dendritic cell; CM, complete medium; DC, dendritic cell;
LCMV, lymphocytic choriomeningitis virus; IL, interleukin.
4 R. Kemp and F. Ronchese, unpublished
observations.
Received 12/ 6/99. Accepted 6/16/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
EL-LCMV (
), LLTC (
), LL-LCMV (
), B16.F10 (
), and
B16.F10GP33 (•) tumor cells were mitomycin C treated and
cultured at the indicated cell number with 5 x 105 T cells from "318" transgenic mice in the presence
of 10 units/ml IL-2. Each data point is the mean from triplicate wells;
bars, SE. B, LLTC (
),
B16.F10GP33 (
