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Department of Microbiology and Carter Immunology Center, University of Virginia, Charlottesville, VA 22908
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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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It has been shown that many Ags derived from MDP have a relatively weak affinity for HLA-A*0201 (5). Low MHC-binding affinity (13) and high MHC dissociation rate (14) of peptides have been correlated with a lack of immunogenicity in generating CD8+ T cell responses in vivo. A high dissociation rate will limit the stable expression of MHC-peptide complexes at the cell surface, to a point at which the activation of naive T cells may be compromised (15). This problem will be particularly acute for immunization strategies that use these MDP-derived epitopes as synthetic peptides. Additionally, because MDP are also expressed in normal melanocytes (8), peripheral tolerance may limit the available CD8+ T cell repertoire (16).4 Therefore, stimulation of therapeutically effective CD8+ T cell responses in melanoma patients will need to address both of these issues.
Dendritic cells (DC) are potent activators of CD8+ T cells and antitumor responses (17), and have been shown to be involved in immune responses elicited by a wide array of immunotherapeutic approaches (18, 19, 20). This has led to the direct use of DC as Ag delivery vehicles in a variety of experimental systems. Ags have been delivered by DC after gene transduction (21, 22) or after endocytosis of particulate preparations or whole proteins (23). In addition, DC have been used to deliver Ags after incubation directly with preprocessed, synthetic peptide Ags (24). In particular, a recent report suggested that DC pulsed with tumor-derived peptides represent an important therapeutic approach to the treatment of melanoma (11). However, DC exist in both immature and mature states that differ in their ability to process and present Ags, as well as in their expression of surface molecules important in T cell activation. The conditions for the optimal use of DC to present various forms of Ag have not been established, and comparisons with other immunization methods have not been systematically made. These issues are difficult to evaluate in the context of clinical trials, and therefore a good animal model is required.
Our laboratory has previously identified several peptide Ags derived from melanoma Ags that are presented by HLA-A*0201 molecules and recognized by CD8+ T cells (8, 25, 26). To gain insight into the use of these peptides for immunotherapy in melanoma patients, we have developed a preclinical model involving transgenic mice that express a recombinant MHC class I molecule in which the peptide binding domain of HLA-A*0201 is combined with the CD8 binding domain of the murine H-2Dd (27). CD8+ T cells from these mice have been shown to recognize HLA-A*0201-restricted Ags that are the same as those recognized by HLA-A*0201+ human CTL (27, 28, 29, 30, 31). In addition, the strength of these responses is comparable with those restricted by endogenous murine H-2 molecules (27). In this study, we report the use of this model system to optimize immunization with DC that have been pulsed with therapeutically relevant HLA-A*0201-restricted melanoma Ags.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synthetic peptides were made by standard Fmoc chemistry using a model AMS422 peptide synthesizer (Gilson, Middleton, WI). All peptides were purified to >98% purity by reverse-phase HPLC on a C-8 column (Vydac, Hesperia, CA). Purity and identity were confirmed using a triple quadropole mass spectrometer (Finnigan, San Jose, CA).
Cell lines
EL4-A2/Kb is a transfectant of the EL4
thymoma that expresses the 
1 + 2 domains of HLA-A*0201 in
association with the 3 domain of H2-Kb (a gift
from Dr. Linda Sherman, The Scripps Institute, San Diego, CA). C1R-AAD
has been previously described (27). Both cell lines were
maintained under selection in RPMI 1640 supplemented with 5%
FBS/SerXtend (Irvine Scientific, Santa Ana, CA) and 300 µg/ml
G418.
HLA-A*0201 peptide-binding assay
This was performed as previously described (32).
Briefly, affinity-purified HLA-A*0201 molecules were incubated at room
temperature with the iodinated indicator peptide, FLPSDYFPSV, and
graded doses of test peptides in PBS, pH 7, containing 0.05% Nonidet
P-40, 1 µM human ß2-microglobulin
(ß2m) (Calbiochem, La Jolla, CA), 1 mM PMSF,
1.3 mM 1,10-phenanthroline, 73 µM pepstatin A, 8 mM EDTA, and 200
µM
N-p-tosyl-L-lysine
chloromethyl ketone (TLCK). After 48 h, class I peptide complexes
were separated from free peptides by gel filtration, and the dose of
individual test peptides that reduced the binding of indicator peptide
by 50% (IC50) was calculated.
Animals
Transgenic mice expressing a chimeric MHC class I composed of
the 1 and 2 domains of HLA-A*0201 and the 3 domain of
H2-Dd (AAD) have been previously described
(27) and were maintained in specific pathogen-free
facilities at the University of Virginia (Charlottesville,
VA).
Dendritic cells
DC were generated as described (33), with
modifications. Bone marrow cells were excised from the femurs and
tibias of AAD+ transgenic mice, depleted of RBC,
and cultured in RPMI 1640 containing 10% FBS, 50 µg/ml gentamicin
(Life Technologies, Grand Island, NY), 7.5 mM HEPES, 2 mM
L-glutamine, 50 µM 2-ME, and 1000 U/ml of both IL-4 and
GM-CSF (generous gifts of Schering-Plough, Kenilworth, NJ). Cultures
were depleted of floating cells and refed with cytokine-supplemented
media after 3 days, and one-half of the medium was replenished after 5
days. After an additional 2–4 days, immature DC (iDC) were isolated by
centrifugation on a 14.5% metrizamide (Sigma, St. Louis, MO) gradient.
Alternatively, iDC from 7-day cultures were then incubated at 5 x
106 cells/well of the above medium, except that
100 U/ml TNF- (Sigma) was substituted for 1000 U/ml IL-4. After 2
days, mature DC (mDC) were isolated on a StemSep column after
incubation with a mixture of Abs for enrichment of mDC (StemCell,
Vancouver, BC, Canada). By immunofluorescence, iDC were characterized
as MHC class Iint, MHC class
IIlow, B7.1low,
B7.2low, and CD40low,
whereas mDC were characterized as MHC class
Ihigh, MHC class IIhigh,
B7.1int, B7.2high, and
CD40int.
Immunization
DC were pulsed with the indicated concentration of peptide for 4 h at 37°C in HBSS containing 5% FBS and 5 µg/ml human ß2m, washed twice, and resuspended in HBSS containing 5% FBS. Mice were injected in tail veins with either 105 DC in 100 µl, or 5 x 107 PFU recombinant vaccinia virus expressing human tyrosinase (28), as indicated.
Ex vivo analysis of activated T cells
CD8+ T cells were enriched from spleens of
immunized mice isolated on a StemSep column after incubation with a
mixture of Abs to enrich for CD8 cells (StemCell). Preparations were
consistently 85–95% CD8+, as assessed by flow
cytometry. These enriched CD8+ T cells were then
directly assessed for either cytolytic activity or cytokine production
using target/stimulator cells that had been pulsed overnight with the
indicated concentration of peptide. For cytotoxicity assays,
peptide-pulsed target cells were labeled with
51Cr and incubated with effector cells for 4
h at 37°C. To measure the production of intracellular cytokines,
peptide-pulsed stimulator cells were incubated with enriched
CD8+ T cells for 5 h at a ratio of 1:1 in
media supplemented with 50 U/ml IL-2 and 5 µg/ml brefeldin A (Sigma).
Stimulated cells were stained with PE-conjugated anti-CD8
(PharMingen, San Diego, CA), washed, fixed, and permeabilized in
PermWash/Fix (PharMingen), and then further stained with
FITC-conjugated anti-IFN-
, anti-TNF-, anti-IL-4,
anti-IL-10 (all from PharMingen), or isotype-matched controls. Flow
cytometry was conducted on a FACScan using CellQuest software. Results
are presented as percentage of positive cells after subtraction of
isotype control values.
In vitro generation of peptide-specific HLA-A*0201-restricted CD8+ T cells
Spleens from primed mice were harvested at least 3 wk after immunization. A total of 1.5 x 107 responder cells and 7 x 106 autologous irradiated (2500 rad) spleen cells that had been pulsed with the indicated concentration of peptide for 3 h at 37°C were incubated in upright 25-cm2 tissue culture flasks (Costar, Cambridge, MA). Alternatively, 5 x 105 residual enriched CD8+ cells from animals immunized 7 days previously and 1 x 107 peptide-pulsed, irradiated autologous splenocytes were incubated in 12-well plates (Costar). After culture for 6–7 days, cytotoxic activity and cytokine production were assessed as described above.
Quantitation of cell surface MHC-peptide complexes
HLA-A*0201+ T2 cells were incubated overnight at 26°C in RPMI 1640 containing 5% FBS to generate a maximal amount of empty but peptide-receptive cell surface HLA-A*0201 molecules. To measure the relative amounts of HLA-A*0201-Tyr369 complexes formed during short-term peptide pulsing, the cells were incubated with the indicated amount of Tyr369 for 3.5 h at 26°C in HBSS supplemented with 5% FBS, 5 µg/ml human ß2m, and 5 µg/ml brefeldin A. The cells were washed twice and incubated for an additional 2 h at 37°C in HBSS supplemented with 5% FBS to allow to determine remaining peptide-free MHC molecules to denature. Alternatively, the relative amounts of HLA-A*0201 complexes formed during short-term peptide pulsing with gp100209, gp100280, or their variants, T2 cells that had been incubated overnight at 26°C were subjected to a mild acid treatment to denature cell surface HLA-A*0201 molecules (34). Acid-stripped cells were then incubated for 3 h at 37°C in 1 ml of RPMI supplemented with 5% FBS and 5 µg/ml ß2m, and the indicated quantity of peptide, or no peptide. In both cases, cells were then washed twice before indirect immunofluorescence staining for HLA-A*0201 expression with the BB7.2 Ab and goat anti-mouse IgG (F(ab')2) FITC (Jackson ImmunoResearch, West Grove, PA), fixed with 1% paraformaldehyde, and analyzed by flow cytometry. Results are expressed as percentage of HLA-A*0201 expression calculated using the formula: [(experimental HLA-A*0201 - no peptide HLA-A*0201)/(untreated T2 HLA-A*0201 at 37°C - no peptide HLA-A*0201)] x 100.
To measure the dissociation rate of peptides from HLA-A*0201, T2 cells that had been incubated overnight at 26°C were loaded with 10 µg/ml of peptide for 20 h at 26°C in RPMI containing 5% FCS and 5 µg/ml ß2m, washed twice, and then incubated at 37°C for the indicated time. Cells were stained and analyzed by flow cytometry, as described above. Results are expressed as geometric mean channel fluorescence after subtraction of the value obtained from cells that had not been incubated with peptide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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iDC are actively phagocytic and efficiently process exogenously
supplied Ags. In contrast, although mDC are inefficient at Ag uptake,
they express higher levels of class I and II MHC and costimulatory
molecules (35). We were interested in whether these two
populations differed in their ability to activate
CD8+ T cells in vivo after they were pulsed with
synthetic peptides. iDC were derived from bone marrow cells of AAD
transgenic mice by culture in GM-CSF and IL-4, and mDC were derived by
further culture of iDC in GM-CSF and TNF-. These two populations
were pulsed with an HLA-A*0201-restricted peptide,
YMDGTMSQV,5 derived
from residues 369–377 of human tyrosinase
(Tyr369), and used to immunize AAD transgenic
mice. After 6 days, splenocytes were isolated, enriched for
CD8+ T cells, and tested immediately for
Ag-specific cytolytic activity. Cells from mice primed with mDC showed
significantly higher lytic activity toward targets pulsed with
Tyr369 than did cells from mice primed with iDC
(Fig. 1
A). This difference was
maintained when splenocytes from mice primed at least 21 days
previously with either iDC or mDC were restimulated in vitro for 1 wk
in the presence of 1 µg/ml Tyr369 (Fig. 1B). These results established that peptide-pulsed mDC are
better than peptide-pulsed iDC in activating CD8+
T cell responses. All additional experiments were thus conducted
using mDC.
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The density of peptide Ag presented on the surface of DC might be
expected to influence the numbers of T cells activated. To address
this, we first established the relationship between the concentration
of peptide used during the pulsing step and the resulting level of
peptide-MHC class I complexes. T2 cells were incubated overnight at
26°C to accumulate peptide-free HLA-A*0201 molecules at the cell
surface and then incubated with increasing concentrations of
Tyr369 for 3.5 h at 26°C. Peptide was
removed and the temperature shifted to 37°C to denature HLA-A*0201
molecules that had not bound peptide. Increasing the peptide
concentration over the range of 0.1–100 µg/ml led to a stepwise
increase in the number of stable HLA-A*0201 molecules, as determined by
indirect immunofluorescence using an HLA-A*0201-specific Ab (Fig. 2A). This indicates that the
epitope density on DC pulsed under comparable conditions should
increase progressively over this concentration range of peptide. We
next asked whether the density of Tyr369 on the
surface of DC used to immunize AAD transgenic mice influenced the
number of activated CD8+ T cells. CD8-enriched
splenocytes were isolated from AAD mice 7 days after immunization with
peptide-pulsed DC and used immediately for measurement of both
Ag-specific cytolytic activity and Ag-induced accumulation of
intracellular IFN-. As the density of
Tyr369-AAD complexes on the DC used for
immunization was increased by changing the pulse concentration from 0.1
to 1 µg/ml, the cytolytic activity and number of IFN--producing
cells also increased (Fig. 2, B and C).
Increasing the pulse concentration from 1 to 10 µg/ml
Tyr369 led to a modest decrease in both
parameters. However, increasing the cell surface density still further
by pulsing DC with 100 µg/ml Tyr369 led to a
significant decrease in both CD8+ cytotoxic
activity and the number of CD8+ IFN--producing
cells. The dose dependence of activation was consistent over several
days, and the highest level of response (as measured by accumulation of
TNF-) was observed on day 7 after immunization, regardless of
peptide dose (Fig. 3). In addition, the
reduction in the response observed at higher peptide doses was not
associated with a shift to a type 2 cytotoxic T cell phenotype, based
on the lack of detectable secretion of either IL-4 or IL-10 (data not
shown). Over the entire dose range, cytotoxic activity roughly
paralleled the number of IFN--producing cells, suggesting that the
major effect was on T cell activation, rather than the nature of the
activated phenotype. These data indicate that the cell surface density
of an epitope presented to a naive T cell population can profoundly
influence the number of CD8+ T cells that
respond, and that the optimum density is less than the maximum
achievable density.
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An important goal of developing a preclinical model is to obtain a
system by which different vaccination protocols can be assessed.
Therefore, we compared the ability of peptide-pulsed mDC and
recombinant vaccinia virus to elicit primary CD8+
T cell responses. We determined that the activity of the primary
CD8+ T cell response to recombinant vaccinia
expressing a minigene encoding Tyr369
(Tyr369-vac) peaked at day 7, and 5 x
107 PFU of recombinant virus was the optimal
immunization dose (data not shown). Thus, AAD mice were primed with
either mDC pulsed with 1 µg/ml Tyr369 or 5
x 107 PFU recombinant
Tyr369-vac. As assessed 7 days after
immunization, mDC stimulated greater ex vivo cytolytic activity on
target cells sensitized with Tyr369 (Fig. 4A). In addition, mDC
stimulated a larger number of peptide-specific
CD8+, as judged by intracellular staining for
IFN- (Fig. 4B). However, after 1 wk of in vitro
restimulation of memory T cell populations, equal levels of lytic
activity were observed from cultures from
Tyr369-vac and mDC-primed mice (Fig. 4C). Therefore, an immunization protocol utilizing this
peptide pulsed onto mDC can elicit CD8+ T cell
responses at least equivalent to a recombinant vaccinia virus encoding
the same peptide.
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We were interested in determining whether other known
HLA-A*0201-restricted peptide Ags derived from MDP also activated
CD8+ T cells in AAD mice, and whether the T cell
responses correlated to peptide dose in a manner similar to
Tyr369. Therefore, we immunized AAD transgenic
mice with mDC that had been pulsed with 1 µg/ml of KTWGQYWQV
(gp100154), ITDQVPFSV
(gp100209), YLEPGPVTA
(gp100280), LLDGTATLRL
(gp100457), or VLYRYGSFSV
(gp100476). We detected weak
CD8+ T cell responses to
gp100154 and gp100476, but
were not able to detect responses to the remaining epitopes either
directly ex vivo after 7 days, or after in vitro restimulation of
splenocytes from animals primed 3 wk previously (Table I). Interestingly, the positive responses
were associated with peptides that had high relative binding affinities
for HLA-A*0201, suggesting that this parameter might be important.
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gp100209(M), in which Met was substituted for Thr
at the P2 anchor position, and gp100280(V), in
which Val was substituted for Ala at the P9 anchor position.
HLA-A*0201-stabilization assays confirmed that these variant peptides
dissociated more slowly from HLA-A*0201 than the parent peptides (Fig. 5A). Because differences in
relative affinity are primarily a function of differences in off rate,
this supports the idea that these substitutions increase the affinity
of the variant peptides relative to the parents. We next determined how
this increase in MHC-binding affinity affected the density of peptides
on the cell surface after short-term peptide pulsing. In agreement with
Fig. 2A, the number of HLA-A*0201 molecules stabilized was
dependent upon the concentration of peptide used (Fig. 5B).
However, at any given concentration, the parent and variant peptides
were equivalent in their ability to stabilize HLA-A*0201 during
short-term incubations of duration equivalent to those used to pulse DC
(Fig. 5B). These results are consistent with the idea that
the number of peptide MHC complexes presented by DC after short-term
incubation with these exogenous peptides is not influenced by
differences in their binding affinities. Instead, it is largely a
function of the on rate, which should be similar or identical for these
peptides. Thus, the effect of amino acid substitutions that increase
binding affinity under these conditions is to prolong epitope
expression on DC, rather than to change the initial level of
expression.
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after 5 h of in vitro culture with cells
that had been pulsed with 100 µg/ml of either peptide. The responses
in mice immunized with DC pulsed with parental
gp100280 were not significantly different from
responses in mice immunized with DC alone, regardless of which peptide
was used for in vitro activation of IFN- accumulation (Fig. 6A). However, substantial
responses were observed in mice immunized with DC that had been pulsed
with gp100280(V). The number of activated
CD8+ T cells increased with the dose of peptide
pulsed onto DC in the range of 0.1–10 µg/ml, consistent with our
earlier observations with Tyr369. In addition,
the highest numbers of CD8+ T cells activated by
gp100280(V) were similar to those observed in
mice immunized with Tyr369-pulsed DC.
Importantly, a fraction of the CD8+ T cells that
were activated by the variant peptide in vivo could recognize the
parent epitope in vitro (Fig. 6A). Similar results were
obtained using DC-pulsed gp100209 or
gp100209(M). Again, no significant response was
observed in mice immunized with DC pulsed with the parent peptide, but
a substantial, dose-dependent response was observed to DC pulsed with
the variant (Fig. 6B). In this case, however, the peak
number of activated CD8+ T cells was consistently
lower than those seen in response to DC pulsed with
Tyr369 or gp100280(V)
(compare Fig. 6B with Figs. 6A and
2C). Again, a proportion of the activated
CD8+ T cells could be stimulated by the parent
peptide. These data confirmed a recent study that used a recombinant
vaccinia virus expressing gp100209(M) to elicit
CD8+ T cell responses to the
gp100209 epitope (38). Collectively,
these results indicate that the introduction of subtle amino acid
substitutions in peptide epitopes, with the intent of decreasing the
dissociation of peptide Ags from the surface of DC, enhances their
immunogenicity.
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, A
and B). Thus, repetitive stimulation with this particular
variant peptide maintained a population of T cells with strong
recognition of the parent peptide. In contrast, when a similar
experiment was conducted using CD8+ T cells from
mice primed with gp100209(M)-pulsed DC, only
cells restimulated with the parent peptide maintained a high level of
recognition (Fig. 7, C and D). Cells that were
maintained on the variant peptide for even 1 wk in vitro showed
substantially lower recognition of the parent. Furthermore, when we
determined the number of peptide-specific T cells in each population
based on accumulation of intracellular IFN-, it was apparent that
only about 30% of cells restimulated with the variant peptide
recognized the parent, as compared with 100% of the cells restimulated
with the parent peptide. The percentage of cross-reactive cells after
in vitro culture with the variant was also lower than that seen in
primary populations (compare Figs. 6B and
8). Taken together, these data suggest
that variant peptides with enhanced MHC-binding affinity are useful for
elicitation of primary immune responses. However, continued stimulation
with some, but not all, variants can lead to the outgrowth of T cells
that have lost reactivity with the parent peptide. The AAD transgenic
mouse model can be used to distinguish these two categories of variant
peptides.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The use of peptide-pulsed DC also allowed us to evaluate how the
surface density of an epitope, in combination with its MHC-binding
affinity, influences the size of the primary CD8+
T cell response in vivo. This issue is not readily addressed using
recombinant viral or bacterial delivery systems. Perhaps not
surprisingly, we found that over a wide dose range, increasing the
density of Ag on the DC resulted in a corresponding increase in the
size of the peptide-specific CD8+ T cell response
until a plateau was reached. It is not yet clear whether this reflects
the preferential activation of only higher avidity T cells at lower Ag
densities, or simply activation of a smaller number of T cells due to a
lower occupancy rate on the DC. We were quite intrigued, however, to
see a substantial decline in the size of the responding population at
the highest Ag density examined. This was observed with several
epitopes. It is possible that at a supraoptimal Ag density, peptide is
shed from the surface of the DC and presented to naive T cells in a
tolerogenic manner. This would be consistent with the observation that
very high doses of peptide in IFA can cause T cell tolerance
(44). On the other hand, at least in vitro, supraoptimal
Ag density can also lead to the apoptosis of high avidity
CD8+ effector T cells via TNF- and
down-regulation of Bcl2 (45).
Although further work will be required to understand the mechanism, it
is apparent that the density of Ag used to activate
CD8+ T cells directly affects the size of the
responding T cell population, and the highest cell surface peptide
density is not necessarily the optimal way of stimulating these
responses. This observation has direct bearing on the use of
peptide-pulsed DC in clinical trial settings.
Our results are consistent with those obtained in a separate study in which recombinant vaccinia viruses were engineered to express different levels of Ag (46). These authors demonstrated that CTL responses increased with increasing Ag expression, but declined at the highest levels of expression. However, our results contrast with another report that demonstrated that the density of Ag above a minimum immunogenic threshold does not influence the size of the CD8+ T cell response (47). In this case, the authors used a recombinant Listeria monocytogenes delivery system and alterations in flanking residues to influence the efficiency of epitope processing and presentation. However, it is uncertain whether the activation of naive CD8+ T cells by microbial immunization is due to the direct infection of professional APC, or results from processing and presentation of Ag after the phagocytosis of infected cells by professional APC. Since Vijh et al. (47) measured the total available peptide on all infected cells, their observations could be a reflection of the amount of Ag that is present in an infected cell at the time it is endocytosed and presented by professional APC (48, 49).
We also used our model system to examine how the immunogenicity of a peptide Ag can be modulated by altering its MHC-binding affinity and dissociation rate. The gp100280 peptide, which has a relatively low HLA-A*0201-binding affinity and fast dissociation rate, was unable to activate CD8+ T cells when presented on DC, even after pulsing with very high concentrations of peptide. In contrast, gp100280(V), which has a relatively higher affinity and slower off rate, activated a substantial CD8+ T cell response against both itself and the parental epitope. This observation is consistent with other studies that have used peptide variants derived from gp100 (50) or Mart-1 (51). However, by using peptide-pulsed DC, we extended this earlier work to establish that the inability of gp100280 to stimulate a response could not be overcome by increasing the pulsing concentration, and thereby the initial level of cell surface epitope expression. In fact, DC expressing a relatively low initial cell surface density of gp100280(V) stimulated significant responses, whereas DC expressing a much higher initial density of gp100280 were nonimmunogenic. The explanation for this paradoxical result is that the short-term pulsing conditions used result in cell surface Ag densities that are largely a function of the peptide on rate, which is usually diffusion controlled and therefore similar or identical for ligands of identical size that are present in solution at the same concentration. However, once exogenous peptide is removed, the cell surface Ag density declines according to its off rate. In the case of gp100280, this decline is apparently so rapid as to render DC pulsed with a very high concentration of this peptide poorly immunogenic.
The mechanism described above may also account for the increased immunogenicity of gp100209(M) compared with nonimmunogenic gp100209. However, immune responses to gp100209 are also likely to be influenced by the fact that this sequence, in contrast to gp100280, is identical in humans and mice. Thus, the gp100209-specific response in AAD transgenic mice may be compromised by one or more mechanisms of self-tolerance. Indeed, gp100209 is nonimmunogenic, despite the fact that its affinity for HLA-A*0201 is similar to that of strongly immunogenic Tyr369. An intriguing possibility is that gp100209, as a self Ag, functions as a partial agonist, whereas gp100209(M) has a sufficiently different conformation that enables it to function as a full agonist (52, 53, 54). It is clear that gp100209(M) induces responses against gp100209, demonstrating that tolerance to the self peptide is partial at best. Again, further investigation into this possible mechanism of activation by variant peptides in the context of tumor Ags that are also self Ags is necessary.
Most immunotherapy protocols involve multiple rounds of immunization to generate large populations of activated tumor-specific CD8+ T cells. We found that CD8+ T cell populations primed and restimulated with gp100280(V) maintained recognition of the parent peptide. A similar result was observed using peptide variants derived from Mart-127–35 (51). Conversely, we found that T cell populations primed and restimulated with gp100209(M) were substantially less effective at recognizing gp100209 than those primed with gp100209(M) and restimulated with the parent epitope. Similar results have been obtained with human PBL from patients immunized with gp100209(M) (55). Although the positions of the substitutions in both of these peptides point down into the HLA-A*0201 binding site, one possible explanation for this difference is that the substitution of Met for Thr at P2 in gp100209 induces a more significant change in the overall conformation of the peptide than the substitution of Val for Ala at P9 in the gp100280 epitope. Alternatively, because the gp100209 sequence is identical in mice and humans, while the gp100280 sequences differ by two residues, endogenous expression of murine gp100209 may lead to more profound tolerance to the human variant peptides used for immunization. If this tolerance is selective for the high avidity, putatively cross-reactive, T cell precursors (56), then the repertoire available for expansion upon immunization with gp100209(M) will be skewed toward lower avidity T cells that are less likely to cross-recognize the parent. Further work will be required to distinguish between these two possibilities.
We have seen recognition of human melanoma cells by some of the T cells
generated against Tyr369 and
gp100209 (data not shown). However, the mice used
for these experiments are transgenic for a chimeric HLA-A*0201, in
which the 3 domain has been substituted with the 3 domain from
H-2Dd. Although this enhances the interaction
between the murine CD8 and the chimeric MHC class I molecule, it
results in a decreased efficiency in recognition of the normal
HLA-A*0201 molecule expressed on human tumor cells. Therefore, even the
failure to recognize a human tumor does not mean that these CTL would
be ineffective against an appropriate AAD+ tumor.
We are currently establishing a model system to evaluate this.
Nonetheless, the results of this study have shown the value of using a
human class I transgenic mouse model to study the variables involved in
immunization with human peptide Ags. We have found that when
peptide-pulsed DC are used for immunization, both the cell surface
density and dissociation rate of the peptide used impact significantly
on the size of the activated population. Although peptide variants with
diminished dissociation rates provide a powerful means to enhance
immunogenicity, our work also suggests that subtle conformational
changes and/or self-tolerance are important issues to be considered in
their use. Transgenic mouse models will be of great use in assessing
such parameters before administration to humans.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Victor H. Engelhard, Department of Microbiology and the Beirne Carter Immunology Center for Immunology Research, University of Virginia Health Sciences Center, MR4 Building, Lane Road, Box 4012, Charlottesville, VA 22908. E-mail address:
3 Abbreviations used in this paper: MDP, melanocyte differentiation protein; ß2m, ß2-microglobulin; DC, dendritic cell; gp, glycoprotein; iDC, immature DC; int, intermediate; mDC, mature DC.
4 T. A. Colella, T. N. J. Bullock, and V. H. Engelhard. Self-tolerance to the murine homologue of a tyrosinase-derived melanoma antigen: implications for tumor immunotherapy. Submitted for publication.
5 The sequence YMDGTMSQV from the tyrosinase protein was initially identified as residues 368–376, and this laboratory had previously used that numbering system (41 ). The correct numbering for the YMDGTMSQV peptide is 369–377.
Received for publication October 12, 1999. Accepted for publication December 17, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 and 3 domains in the recognition of self and non-self MHC molecules. J. Immunol. 156:2473.[Abstract]
receptor II-mediated apoptosis of cytotoxic T lymphocytes. J. Exp. Med. 188:1391.This article has been cited by other articles:
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  C. C. Brinkman, S. L. Sheasley-O'Neill, A. R. Ferguson, and V. H. Engelhard Activated CD8 T Cells Redistribute to Antigen-Free Lymph Nodes and Exhibit Effector and Memory Characteristics J. Immunol., August 1, 2008; 181(3): 1814 - 1824. [Abstract] [Full Text] [PDF]
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M. J. Smithey, S. Brandt, N. E. Freitag, D. E. Higgins, and H. G. A. Bouwer Stimulation of Enhanced CD8 T Cell Responses Following Immunization with a Hyper-Antigen Secreting Intracytosolic Bacterial Pathogen J. Immunol., March 1, 2008; 180(5): 3406 - 3416. [Abstract] [Full Text] [PDF]
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M. L. Hwang, J. R. Lukens, and T. N. J. Bullock Cognate Memory CD4+ T Cells Generated with Dendritic Cell Priming Influence the Expansion, Trafficking, and Differentiation of Secondary CD8+ T Cells and Enhance Tumor Control J. Immunol., November 1, 2007; 179(9): 5829 - 5838. [Abstract] [Full Text] [PDF]
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 D.-W. Liu, Y.-C. Yang, H.-F. Lin, M.-F. Lin, Y.-W. Cheng, C.-C. Chu, Y.-P. Tsao, and S.-L. Chen Cytotoxic T-Lymphocyte Responses to Human Papillomavirus Type 16 E5 and E7 Proteins and HLA-A*0201-Restricted T-Cell Peptides in Cervical Cancer Patients J. Virol., March 15, 2007; 81(6): 2869 - 2879. [Abstract] [Full Text] [PDF]
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S. L. Sheasley-O'Neill, C. C. Brinkman, A. R. Ferguson, M. C. Dispenza, and V. H. Engelhard Dendritic Cell Immunization Route Determines Integrin Expression and Lymphoid and Nonlymphoid Tissue Distribution of CD8 T Cells J. Immunol., February 1, 2007; 178(3): 1512 - 1522. [Abstract] [Full Text] [PDF]
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K. M. Hargadon, C. C. Brinkman, S. L. Sheasley-O'Neill, L. A. Nichols, T. N. J. Bullock, and V. H. Engelhard Incomplete Differentiation of Antigen-Specific CD8 T Cells in Tumor-Draining Lymph Nodes J. Immunol., November 1, 2006; 177(9): 6081 - 6090. [Abstract] [Full Text] [PDF]
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 A. D. Gritzapis, L. G. Mahaira, S. A. Perez, N. T. Cacoullos, M. Papamichail, and C. N. Baxevanis Vaccination with Human HER-2/neu (435-443) CTL Peptide Induces Effective Antitumor Immunity against HER-2/neu-Expressing Tumor Cells In vivo. Cancer Res., May 15, 2006; 66(10): 5452 - 5460. [Abstract] [Full Text] [PDF]
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D. W. Mullins and V. H. Engelhard Limited infiltration of exogenous dendritic cells and naive T cells restricts immune responses in peripheral lymph nodes. J. Immunol., April 15, 2006; 176(8): 4535 - 4542. [Abstract] [Full Text] [PDF]
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A. Margalit, H. M. Sheikhet, Y. Carmi, D. Berko, E. Tzehoval, L. Eisenbach, and G. Gross Induction of Antitumor Immunity by CTL Epitopes Genetically Linked to Membrane-Anchored {beta}2-Microglobulin J. Immunol., January 1, 2006; 176(1): 217 - 224. [Abstract] [Full Text] [PDF]
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 P. M. Lavoie, A. R. Dumont, H. McGrath, A.-E. Kernaleguen, and R.-P. Sekaly Delayed expansion of a restricted T cell repertoire by low-density TCR ligands Int. Immunol., July 1, 2005; 17(7): 931 - 941. [Abstract] [Full Text] [PDF]
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D. Dieckmann, E. S. Schultz, B. Ring, P. Chames, G. Held, H. R. Hoogenboom, and G. Schuler Optimizing the exogenous antigen loading of monocyte-derived dendritic cells Int. Immunol., May 1, 2005; 17(5): 621 - 635. [Abstract] [Full Text] [PDF]
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T. N. J. Bullock and H. Yagita Induction of CD70 on Dendritic Cells through CD40 or TLR Stimulation Contributes to the Development of CD8+ T Cell Responses in the Absence of CD4+ T Cells J. Immunol., January 15, 2005; 174(2): 710 - 717. [Abstract] [Full Text] [PDF]
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R. A. Kemp, T. J. Powell, D. W. Dwyer, and R. W. Dutton Cutting Edge: Regulation of CD8+ T Cell Effector Population Size J. Immunol., September 1, 2004; 173(5): 2923 - 2927. [Abstract] [Full Text] [PDF]
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S. B. J. Wong, C. B. Buck, X. Shen, and R. F. Siliciano An Evaluation of Enforced Rapid Proteasomal Degradation as a Means of Enhancing Vaccine-Induced CTL Responses J. Immunol., September 1, 2004; 173(5): 3073 - 3083. [Abstract] [Full Text] [PDF]
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Y. Yoshimura, R. Yadav, G. J. Christianson, W. U. Ajayi, D. C. Roopenian, and S. Joyce Duration of Alloantigen Presentation and Avidity of T Cell Antigen Recognition Correlate with Immunodominance of CTL Response to Minor Histocompatibility Antigens J. Immunol., June 1, 2004; 172(11): 6666 - 6674. [Abstract] [Full Text] [PDF]
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 D. W. Mullins, S. L. Sheasley, R. M. Ream, T. N.J. Bullock, Y.-X. Fu, and V. H. Engelhard Route of Immunization with Peptide-pulsed Dendritic Cells Controls the Distribution of Memory and Effector T Cells in Lymphoid Tissues and Determines the Pattern of Regional Tumor Control J. Exp. Med., October 6, 2003; 198(7): 1023 - 1034. [Abstract] [Full Text] [PDF]
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T. N. J. Bullock, D. W. Mullins, and V. H. Engelhard Antigen Density Presented By Dendritic Cells In Vivo Differentially Affects the Number and Avidity of Primary, Memory, and Recall CD8+ T Cells J. Immunol., February 15, 2003; 170(4): 1822 - 1829. [Abstract] [Full Text] [PDF]
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P. Luhrs, W. Schmidt, R. Kutil, M. Buschle, S. N. Wagner, G. Stingl, and A. Schneeberger Induction of Specific Immune Responses by Polycation-Based Vaccines J. Immunol., November 1, 2002; 169(9): 5217 - 5226. [Abstract] [Full Text] [PDF]
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G. L. Eiben, M. P. Velders, H. Schreiber, M. C. Cassetti, J. K. Pullen, L. R. Smith, and W. M. Kast Establishment of an HLA-A*0201 Human Papillomavirus Type 16 Tumor Model to Determine the Efficacy of Vaccination Strategies in HLA-A*0201 Transgenic Mice Cancer Res., October 15, 2002; 62(20): 5792 - 5799. [Abstract] [Full Text] [PDF]
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E. J. Wherry, M. J. McElhaugh, and L. C. Eisenlohr Generation of CD8+ T Cell Memory in Response to Low, High, and Excessive Levels of Epitope J. Immunol., May 1, 2002; 168(9): 4455 - 4461. [Abstract] [Full Text] [PDF]
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T. N. J. Bullock, D. W. Mullins, T. A. Colella, and V. H. Engelhard Manipulation of Avidity to Improve Effectiveness of Adoptively Transferred CD8+ T Cells for Melanoma Immunotherapy in Human MHC Class I-Transgenic Mice J. Immunol., November 15, 2001; 167(10): 5824 - 5831. [Abstract] [Full Text] [PDF]
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J.-S. Blanchet, D. Valmori, I. Dufau, M. Ayyoub, C. Nguyen, P. Guillaume, B. Monsarrat, J.-C. Cerottini, P. Romero, and J. E. Gairin A New Generation of Melan-A/MART-1 Peptides That Fulfill Both Increased Immunogenicity and High Resistance to Biodegradation: Implication for Molecular Anti-Melanoma Immunotherapy J. Immunol., November 15, 2001; 167(10): 5852 - 5861. [Abstract] [Full Text] [PDF]
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D. W. Mullins, T. N. J. Bullock, T. A. Colella, V. V. Robila, and V. H. Engelhard Immune Responses to the HLA-A*0201-Restricted Epitopes of Tyrosinase and Glycoprotein 100 Enable Control of Melanoma Outgrowth in HLA-A*0201-Transgenic Mice J. Immunol., November 1, 2001; 167(9): 4853 - 4860. [Abstract] [Full Text] [PDF]
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R. A. Pierce, E. D. Field, T. Mutis, T. N. Golovina, C. V. Kap-Herr, M. Wilke., J. Pool, J. Shabanowitz, M. J. Pettenati, L. C. Eisenlohr, et al. The HA-2 Minor Histocompatibility Antigen Is Derived from a Diallelic Gene Encoding a Novel Human Class I Myosin Protein J. Immunol., September 15, 2001; 167(6): 3223 - 3230. [Abstract] [Full Text] [PDF]
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S. Pascolo, M. Schirle, B. Gückel, T. Dumrese, S. Stumm, S. Kayser, A. Moris, D. Wallwiener, H.-G. Rammensee, and S. Stevanovic A MAGE-A1 HLA-A*0201 Epitope Identified by Mass Spectrometry Cancer Res., May 1, 2001; 61(10): 4072 - 4077. [Abstract] [Full Text]
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K. Shimizu, E. K. Thomas, M. Giedlin, and J. J. Mulé Enhancement of Tumor Lysate- and Peptide-pulsed Dendritic Cell-based Vaccines by the Addition of Foreign Helper Protein Cancer Res., March 1, 2001; 61(6): 2618 - 2624. [Abstract] [Full Text]
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D. R. Siemens, B. D. Elzey, D. M. Lubaroff, C. Bohlken, R. J. Jensen, A. K. Swanson, and T. L. Ratliff Cutting Edge: Restoration of the Ability to Generate CTL in Mice Immune to Adenovirus by Delivery of Virus in a Collagen-Based Matrix J. Immunol., January 15, 2001; 166(2): 731 - 735. [Abstract] [Full Text] [PDF]
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A. L. Zarling, S. B. Ficarro, F. M. White, J. Shabanowitz, D. F. Hunt, and V. H. Engelhard Phosphorylated Peptides Are Naturally Processed and Presented by Major Histocompatibility Complex Class I Molecules in Vivo J. Exp. Med., December 18, 2000; 192(12): 1755 - 1762. [Abstract] [Full Text] [PDF]
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M. Larsson, D. Messmer, S. Somersan, J.-F. Fonteneau, S. M. Donahoe, M. Lee, P. R. Dunbar, V. Cerundolo, I. Julkunen, D. F. Nixon, et al. Requirement of Mature Dendritic Cells for Efficient Activation of Influenza A-Specific Memory CD8+ T Cells J. Immunol., August 1, 2000; 165(3): 1182 - 1190. [Abstract] [Full Text] [PDF]
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V. L. Crotzer, R. E. Christian, J. M. Brooks, J. Shabanowitz, R. E. Settlage, J. A. Marto, F. M. White, A. B. Rickinson, D. F. Hunt, and V. H. Engelhard Immunodominance Among EBV-Derived Epitopes Restricted by HLA-B27 Does Not Correlate with Epitope Abundance in EBV-Transformed B-Lymphoblastoid Cell Lines J. Immunol., June 15, 2000; 164(12): 6120 - 6129. [Abstract] [Full Text] [PDF]
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T. A. Colella, T. N.J. Bullock, L. B. Russell, D. W. Mullins, W. W. Overwijk, C. J. Luckey, R. A. Pierce, N. P. Restifo, and V. H. Engelhard Self-Tolerance to the Murine Homologue of a Tyrosinase-Derived Melanoma Antigen: Implications for Tumor Immunotherapy J. Exp. Med., April 3, 2000; 191(7): 1221 - 1232. [Abstract] [Full Text] [PDF]
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, 
) or immature
(
, 
) DC that had been pulsed with 1 µg/ml Tyr369
peptide. A, Spleens were removed 6 days after
immunization. Splenocytes were enriched for CD8+ T cells,
as described in Materials and Methods, and assayed on
51Cr-labeled EL4-AAK prepulsed with 10 µg/ml
Tyr369 peptide (filled symbols), or left unpulsed (open
symbols). B, Splenocytes from mice primed 
21 days
previously with DC pulsed with 1 µg/ml Tyr369 were
restimulated in vitro for 6 days with irradiated autologous splenocytes
that had been pulsed with 1 µg/ml Tyr369.
) or left unpulsed (
).
C, Accumulation of intracellular IFN-
), or 1 (
) µg/ml of Tyr369, and
spleens were harvested after the indicated number of days. Enriched
CD8+ T cells were incubated with EL4-AAK stimulators that
had been pulsed with 100 µg/ml Tyr369 (filled symbols),
or left unpulsed (open symbols), and then assayed for intracellular
TNF-
, gp100209
variant), according to the protocol described in the Materials
and Methods, before staining for HLA-A*0201.
