From the Institute for Medical Microbiology and Hygiene, Department of Immunology, University of Freiburg, 79104 Freiburg, Germany and the § Institut de Biologie Moléculaire et Cellulaire, UPR 9021 CNRS, 67000 Strasbourg, France
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Recent investigations have suggested that
pseudopeptides containing modified peptide bonds might advantageously
replace natural peptides in therapeutic strategies. We have generated
eight reduced peptide bond T cells recognize antigenic peptides in association with
MHC1 molecules and play a key
role in protection against harmful pathogens and in tumor elimination.
Over the past years many attempts have been made to use synthetic
peptides as potential vaccines and immunoregulatory agents (1, 2).
Recent studies have provided evidence that pseudopeptides, in which one
or several of the natural amide bonds (CO-NH) are replaced by CO-NH
isosters (3), can have enhanced antigenic and immunogenic properties
(4-6). Most interestingly, it has been shown that such peptide
analogues can bind to class I and II MHC molecules (7-13), and some of
them also induce differential effects on T cell responsiveness (14) similar to those described with altered peptide ligands, which contain
single amino acid replacements (15). These several recent studies thus
demonstrated the potential interest of pseudopeptides as possible
therapeutic strategies in T cell-mediated disorders.
The high susceptibility of synthetic peptides to proteases is
considered to be a major drawback for their use as vaccines or
immunoregulatory molecules (16). We have previously shown that several
pseudopeptides with enhanced antigenic activity are more resistant to
proteolytic degradation in vitro (5, 6, 14, 17). However, no
information on in vivo stability of such peptide analogues
and the possible direct contribution of this resistance in their
ability to modulate the immune response is available. Increased
biological activity of protease-resistant pseudopeptide analogues has
already been widely shown in other fields of medical chemistry (for
review, see Ref. 18, 19-22). The retro-inverso analogue of the
immunostimulatory molecule tuftsin is an outstanding example of a
bioactive tetrapeptide for which biological efficiency in
vivo has been remarkably increased by introducing one modified
peptide bond (22). To examine the possible influence of proteolytic
resistance of MHC class I binding peptides on their biological
activity, we designed a series of eight reduced peptide bond
pseudopeptides of the immunodominant CD8+ T cell epitope of
LCMV in C57BL/6 (B6, H-2b) mice. This CTL epitope (called
GP33) is located in residues 33-41 of the LCMV glycoprotein (23). We
tested the capacity of these eight analogues, each containing one
reduced peptide bond Peptides--
LCMV glycoprotein 33-41 peptide (GP33, KAVYNFATM)
(23) and the control Db-restricted adenovirus peptide
234-243 (E1A, SGPSNTPPEI) (24) were purchased from Neosystem
(Strasbourg, France). To prevent dimer formation, the original cysteine
residue present at the anchor position 41 of the GP33 peptide was
replaced by a methionine residue. The reduced peptide bond analogues
were synthesized, purified, and analyzed as described previously
(25).
Protease Resistance Analysis in Vitro and in Vivo--
To study
the resistance of GP33 and Mice and Virus--
B6 mice were obtained from our breeding
colony (Institute for Medical Microbiology and Hygiene) and from Harlan
(Borchen, Germany). The TCR Tg mouse (LCMV TCR+ mouse)
expressing a TCR specific for the immunodominant epitope GP33 in a
Db restriction context (line 318) has been described
previously (26). Animals were kept under conventional conditions and
used 12-24 weeks after birth. For immunizations, peptides were
dissolved in PBS and in some experiments emulsified 1:1 (v/v) in IFA.
Frequency, route of injection, and amounts of peptide are specified in
the figure legends and in the text. The LCMV-WE virus strain used in
this study was originally obtained from F. Lehmann-Grube and was
propagated on L929 fibroblast cells. Mice were injected intravenously with 200 plaque-forming units of LCMV-WE, and the virus titer in the
spleen was determined 4 days later as described previously (27).
Proliferation and Cytotoxicity Assays--
In proliferation
assays, responder spleen cells from LCMV TCR+ mice were
incubated in 96-well plates (4 × 105 cells/well) with
serial dilutions of the various peptides. Forty-eight hours later,
cultures were pulsed with 1 µCi/well [3H]thymidine for
8 h and harvested onto filter papers. Results are displayed as cpm
determined with a Trace 96 counter (Berthold-Inotech, Jaffrey, NH).
Cytolytic activity was determined in a 51Cr release assay
as described (28), using as target cells 51Cr-labeled EL4
coated with decreasing concentrations (10 MHC-Peptide Complex Stabilization Assay--
The binding of GP33
and peptide analogues to MHC molecules was assessed by the
stabilization assay using transporter associated with antigen
processing-deficient RMA-S cells (29). RMA-S cells (H-2b)
were cultured overnight at 25 °C to achieve maximal expression of
empty MHC molecules on the cell surface. Cells (5 × 105) were then incubated for 4 h at 37 °C in
96-well round-bottom plates with different peptide concentrations.
Cells were then washed once and stained for Db expression
with the anti-Db monoclonal antibody B-22-243 (30) followed
by fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Caltag,
San Francisco, CA). The quantity of stabilized Db molecules
on the cell surface was determined by flow cytometry using a FACSort
flow cytometer (Becton-Dickinson, Montain View, CA).
Binding of GP33 Analogues to H-2Db MHC
Molecules--
Eight reduced peptide bond pseudopeptides corresponding
to the H-2Db-restricted CD8+ T cell GP33 LCMV
epitope were used in this study. These analogues were obtained by
replacing one natural peptide bond at a time by a reduced peptide bond
The Stability to Proteases of the GP33 Parent and The In Vivo Half-life of the Peptide-MHC Complexes--
The in
vivo half-life of the peptide-MHC complexes was determined on
spleen cells from mice injected with either the parent GP33 peptide or
the The The use of peptides corresponding to MHC class I epitopes to
induce a protective CTL response against viruses or tumors is of
particular interest in the development of peptide-based vaccines. Recent investigations have suggested that antigenic pseudopeptides containing one or several peptide bond isosters might advantageously replace natural peptides in therapeutic strategies because they can
bind to MHC class I molecules and generate an efficient T cell response
(7, 13, 14). In this study, we have examined the antigenic and
immunogenic properties of pseudopeptide analogues derived from the LCMV
glycoprotein peptide 33-41 (GP33) in which one CO-NH amide bond at a
time was replaced by a reduced peptide bond Two of eight analogues studied, namely CD8+ T cells from LCMV TCR+ mice recognized
equally well the parent GP33 peptide and the Because the parent and In good agreement with previous results (35, 36), the estimated
functional half-life of complexes formed in vivo by
H-2Db and GP33 was ~10-15 h. In the same test, the
half-life of the Because of the fact that GP33 and (CH2-NH) analogues
corresponding to the H-2Db-restricted CD8+ T
cell epitope (called GP33) of the glycoprotein of the lymphocytic choriomeningitis virus. One of these pseudopeptides, containing a
reduced peptide bond between residues 6 and 7 (
(6-7)), displayed very similar properties of binding to major histocompatibility complex
(MHC) and recognition by T cell receptor transgenic T cells specific
for GP33 when compared with the parent peptide. We assessed in
vitro and in vivo the proteolytic resistance of GP33
and
(6-7) and analyzed its contribution to the priming properties of these peptides. The
(6-7) analogue exhibited a dramatically increased proteolytic resistance when compared with GP33, and we show
for the first time that MHC-peptide complexes formed in vivo with a pseudopeptide display a sustained half-life compared with the complexes formed with the natural peptide. Furthermore, in
contrast to immunizations with GP33, three injections of
(6-7) in
saline induced significant antiviral protection in mice. The enhanced
ability of
(6-7) to induce antiviral protection may result from the
higher stability of the analogue and/or of the MHC-analogue complexes.
INTRODUCTION
Top
Abstract
Introduction
References
(CH2-NH) at successive positions,
to bind to H-2Db MHC molecules. The peptides that bound
significantly to H-2Db were further analyzed with respect
to their recognition by GP33-specific T cells from a Tg mouse that
expresses a H-2Db-restricted T cell receptor specific for
this epitope and for their ability to induce antiviral protection.
MATERIALS AND METHODS
(6-7) peptides to proteases in
vitro, peptides (625 µg/ml) were incubated at 20 °C in fresh
mouse serum diluted two times in PBS, pH 7.4. The reaction was stopped
at intervals by adding trifluoroacetic acid (10% of the final volume).
The suspension was diluted five times with water and centrifuged at
2000 × g. The extent of peptide cleavage was estimated
by reversed phase HPLC as described previously (6), and the main
cleavage site was identified by mass analysis of the compounds present
in the major HPLC peaks after a 1-min incubation in fresh mouse serum.
To study the protease resistance of peptide in vivo, mice
were injected intravenously with 200 µg of peptide in 400 µl of
PBS. Blood was collected at intervals from retroorbital venus plexus,
and serum samples were prepared and immediately stored at
20 °C
until use. The presence of remaining GP33 and
(6-7) peptides in
sera was assessed using a proliferation assay with LCMV
TCR+ T cells (see below) using the sera diluted
1:100-1:3000. To analyze the presence of peptides on spleen cells, the
mice were injected intravenously with peptide, and after the indicated
time points, spleen cells were isolated and used as stimulators (2 × 105 cells/well) in a proliferation assay with LCMV
TCR+ T cells. The percentage of proliferation was
calculated using the following ratio: (cpm obtained with serum or
APC/cpm obtained with exogenously added 10
7 M
peptide) × 100. The proliferation level obtained with
10
7 M GP33 peptide was determined in each experiment.
6 to
10
10 M) of the different peptides and as
responder cells Tg effector cells at the indicated effector-to-target
ratios. The responder LCMV TCR+ effector T cells were
generated by co-culturing 4 × 106 spleen cells from
LCMV TCR+ mice for 3 days before the CTL assay with 2 × 106 GP33-loaded B6 spleen cells.
RESULTS
(CH2-NH). The amino acid sequence and characteristics
of these peptides are shown in Table I.
The eight analogues were first tested for their ability to bind to MHC
Db class I molecules using transporter associated with
antigen processing-deficient RMA-S cells. The stabilized
Db-peptide complexes were quantified by flow cytometry
using a Db-specific monoclonal antibody. Two of the eight
pseudopeptides, namely those containing a reduced peptide bond between
residues Asn5 and Phe6, analogue
(5-6), and
between Phe6 and Ala7, analogue
(6-7),
bound to Db molecules as efficiently as the parent GP33
peptide (Fig. 1). The six other analogues
were unable to stabilize Db molecules at a physiological
range of peptide concentration.
Peptide sequence, retention times (Rt) in analytical reversed phase
HPLC, and peptide masses of the GP33 peptide and reduced peptide bond
analogues
View larger version (25K):
[in a new window]
Fig. 1.
Binding of GP33 analogues to
H-2Db MHC molecules. Parent GP33 peptide and the GP33
pseudopeptides were tested at different concentrations
(10 4 to 10
10 M) to stabilize
MHC class I H-2Db molecules present on the surface of RMA-S
cells. Stabilization of molecules was determined by flow cytometry
using the Db-specific monoclonal antibody B-22-243.
(6-7) Analogue Is Recognized by T Cells from LCMV
TCR+ Mice as Efficiently as the GP33 Parent
Peptide--
Proliferation assays with CD8+ T cells from
LCMV TCR+ mice were used to examine T cell recognition of
the two Db binding analogues
(5-6) and
(6-7). As
shown in Fig. 2A, the
(5-6) analogue did not stimulate LCMV TCR+ T cells. In
contrast, the
(6-7) analogue induced proliferation of Tg T cells as
efficiently as the GP33 peptide. Peptide recognition by LCMV
TCR+ effector T cells was further examined in
51Cr release assays using EL-4 target cells loaded with
different concentrations of GP33
(5-6) and
(6-7) peptides and
the control Db-restricted adenovirus E1A peptide. The Tg
effector T cells, generated in vitro in the presence of
GP33-loaded APC, lysed target cells presenting GP33 and
(6-7)
peptides to a similar extent but failed to recognize target cells
loaded with the
(5-6) peptide and control E1A peptide (Fig.
2B). Taken together, these data clearly show that the
(6-7) analogue is recognized as efficiently as the parent GP33
peptide by LCMV TCR+ T cells. For further analysis, only
the
(6-7) analogue was used.
View larger version (20K):
[in a new window]
Fig. 2.
LCMV TCR+ T cells efficiently
recognize the (6-7) but not the
(5-6) analogue. A,
proliferation of LCMV TCR+ spleen cells in the presence of
the indicated concentrations of peptide measured by
[3H]thymidine incorporation. B, specific lysis
by LCMV TCR+ effector cells of EL4 target cells loaded with
10
6, 10
8, or 10
10
M peptides GP33
(5-6) and
(6-7) and the control
peptide E1A. The E1A peptide is a Db binding epitope from
adenovirus and was used as a negative control.
(6-7) Peptides in
Vitro--
The proteolytic degradation of the GP33 and
(6-7)
peptides by proteases present in mouse serum was examined by HPLC. The half-life of the
(6-7) peptide in serum was superior to 1 h, whereas the parent peptide GP33 was almost completely degraded within a
few minutes (Fig. 3A). When
the GP33 peptide was analyzed by HPLC shortly after digestion (1 min),
two main peaks were observed (Fig. 3B). Mass spectrometry
analysis revealed that the complete GP33 peptide of sequence KAVYNFATM
was present in peak 1, whereas a peptide fragment corresponding to the
6 N-terminal residues (KAVYNF) was eluted in peak 2. These results
suggest that the GP33 peptide contains a particularly sensitive site
between residues 6 and 7 that is rapidly cleaved by mouse serum
proteases in vitro and that this site is probably
"protected" from cleavage in the remarkably more resistant
(6-7) analogue.
View larger version (19K):
[in a new window]
Fig. 3.
The (6-7) analogue is highly resistant to
in vitro protease digestion, and the major proteolytic
cleavage site in the GP33 parent peptide is located between residues 6 and 7. A, proteolytic degradation of GP33 and
(6-7)
peptides in mouse serum. At the time points indicated, the digestion
was stopped, and the remaining peptide fragments were analyzed by HPLC
as described under "Materials and Methods." B,
determination of the cleavage site in the GP33 parent peptide after
digestion by proteolytic enzymes. At time points 0 and 1 min of
incubation of the GP33 parent peptide with mouse serum, the main peaks
eluted from the HPLC column were analyzed by mass spectrometry. The
cleavage site was deduced from the mass of the main fragment appearing
after 1 min of digestion (Peak 2). Peak 1 corresponds to undigested GP33 peptide.
(6-7) Analogue Exhibits a Longer Half-life in Vivo--
To
measure the half-life of the GP33 and
(6-7) peptides in
vivo, B6 mice were injected intravenously with peptides, and the peptide remaining over the time in the serum of these animals was
determined using antigen-induced proliferation of LCMV TCR+
T cells as an experimental readout. Using the mouse sera diluted 1:100
in this very sensitive functional test, the presence of GP33 peptide
could not be detected in blood 5 min after peptide injection (Fig.
4, A and B). In
contrast,
(6-7) peptide was readily detectable after 5 min using
1:3000-fold diluted sera from mice injected with the analogue (Fig.
4A). Using the sera diluted 1:100, the presence of the
analogue was still clearly measurable 1 h after injection of mice
(Fig. 4B).
View larger version (25K):
[in a new window]
Fig. 4.
The (6-7) analogue persists longer than
the parent peptide both in the serum and on the spleen cells of
peptide-injected B6 mice. For experiments described in
A-C, mice were injected one time intravenously in the tail
vein with 200 µg of GP33 or
(6-7) peptides. A, 5 min
after peptide injection, serum was taken from the retroorbital venus
plexus and incubated with LCMV TCR+ cells in a
proliferation assay. At dilutions <1:100, mouse serum was toxic for
the cultures. B, pharmacokinetics of the
(6-7) analogue
and the GP33 peptide in the serum. At the indicated time points after
peptide injection, serum was taken and incubated at a 1:100 dilution
with LCMV TCR+ cells in a proliferation assay.
C, sustained presence of the
(6-7) analogue on spleen
cells. At the indicated time points after peptide injection, mice were
killed, and splenocytes were used as stimulators in a proliferation
assay with LCMV TCR+ T cells. The data in A-C
correspond to the mean obtained from experimental groups of at least
three mice.
(6-7) analogue. In these experiments, spleen cells from
peptide-injected B6 mice were directly used as APC for LCMV
TCR+ T cells in a proliferation assay. As shown in Fig.
4C, the spleen cells were rapidly loaded with both GP33 and
(6-7) peptides (~10 min after injection), and the stimulatory
capacities of spleen cells remained comparable for both peptides until
~15 h. However, 24-30 h after peptide injection, only spleen cells
from
(6-7) peptide-injected mice were found to be stimulatory for
LCMV TCR+ T cells. Thus, the half-life of the
(6-7)
peptide-Db complex in vivo on spleen cells was
~2-fold increased when compared with the parent GP33 peptide (30-40
versus 15-24 h).
(6-7) Peptide Analogue Efficiently Induces Antiviral
Protection--
The data described above show that as efficiently as
the parent peptide GP33, the
(6-7) analogue is recognized by the Tg LCMV TCR and that the half-life of the
(6-7) peptide-Db
complex in vivo is significantly increased compared with
that of GP33 peptide-Db complex. To further examine the
potential advantage of using the
(6-7) peptide in vivo,
we tested the ability of the analogue to induce antiviral protection.
Because of the high frequency of GP33-specific T cells in LCMV
TCR+ mice, antiviral protection induced by peptides cannot
be directly examined in these mice, because inoculated virus is rapidly
cleared (23). Therefore, we examined antiviral protection in B6 mice in
which a small number (105/mouse) of LCMV TCR+ T
cells were adoptively transferred. After peptide immunization, mice
were challenged with LCMV and virus titers were determined as described
under "Materials and Methods." As shown in Fig.
5A, peptide immunization using
subcutaneous injection of either the GP33 parent peptide or
(6-7)
analogue in IFA induced a similar extent of antiviral protection. On
the other hand, no significant protection was observed when mice
received a single subcutaneous injection of 100 µg of GP33 or
(6-7) peptides in the absence of IFA (Fig. 5B). However,
when mice received three successive subcutaneous injections of 50 µg
of peptides in the absence of IFA (Fig. 5B), a significant
decrease (10-100-fold) of virus titer was observed in mice immunized
with
(6-7), whereas no effect was found in mice immunized with
the GP33 parent peptide.
View larger version (20K):
[in a new window]
Fig. 5.
Antiviral protection induced by GP33 and
(6-7) peptides. One day before peptide immunization, LCMV
TCR+ naive T cells (105 cells/mouse in 500 µl
of PBS) were transferred into nontransgenic B6 mice. Ten days after
immunizations, mice were challenged with LCMV, and 4 days later, virus
titers in the spleens were determined. Dots represent
individual mice (at least three per group). The full lines
are the mean of all values within each experimental group. The
dotted line corresponds to the detection limit of the virus
plaque assay. A, peptides injected subcutaneously at the
indicated doses in the presence of IFA. B, peptides injected
in PBS using the indicated routes and frequency of injection.
DISCUSSION
(CH2-NH) in
the native sequence. GP33 is presented by H-2Db MHC
molecules and recognized in this context by specific CD8+ T
cells. This model was particularly interesting for at least three
reasons. First, although infection with cytopathic viruses (e.g. vaccina, vesicular stomatis, Semliki Forest, or
influenza virus) is controlled by soluble mediators such as antibodies
and cytokines, T cell-mediated cytotoxicity is crucial for the
resolution of infections with noncytopathic viruses such as LCMV (31). LCMV is thus an excellent model to study in vivo the
efficacy of the CTL response induced by modified peptides. Second, it
is known that although the parent peptide GP33 is particularly
efficient to induce protection against a viral challenge when it is
injected in the presence of IFA, this peptide is unable to generate
protection when used in saline solution. Third, a transgenic model
containing within the CD8+ T cell population 40-60%
transgenic TCR+ (V
2/V
8) T cells specific for peptide
GP33 presented in the H-2Db MHC context is available (27),
thus allowing in vivo study of the recognition of the
peptide (or pseudopeptide)-MHC complexes by this TCR.
(5-6) and
(6-7), were
able to bind to H-2Db molecules with an apparent affinity
similar to that of the parent GP33 peptide. Guichard et al.
(7) previously found that five of eight reduced peptide bond analogues
derived from a Plasmodium berghei MHC class I epitope could
bind to soluble recombinant H-2Kd molecules. However, the
relative affinity of these pseudopeptides to MHC molecules was
5-10-fold lower than that of the parent peptide. The present finding
that most of the reduced peptide bond analogues of GP33 exhibited a
decreased or no MHC binding capacity correlates with crystallographic
data indicating that the peptide backbone plays an important role for
binding of peptide to MHC class I molecules (32). It also suggests that
in this case, the carbonyl oxygens of the residues in positions 5 and 6 are not essential for peptide-MHC binding.
(6-7) analogue. In
contrast, they did not recognize the
(5-6) analogue presented in
the H-2Db context. This result suggests either that the
carbonyl oxygen of the residue in position 5 is directly involved in
the interaction with the TCR or that this oxygen atom influences the
orientation of the Phe6 side chain that has been shown to
be crucial for TCR recognition (23, 33, 34).
(6-7) peptides share similar antigenic
properties, the role of their respective susceptibility to proteases in
relation to their biological activity could be investigated. We found
that the level of GP33 resistance to mouse proteases drastically
increased when a single peptide bond located between residues 6 and 7 was replaced by a reduced peptide bond in analogue
(6-7). This
result fits well with the observation that a highly protease-sensitive
cleavage site is located between positions 6 and 7 in GP33. The
detailed molecular mechanisms of GP33 degradation have not yet been
elucidated. Cleavage by an endopeptidase remains the most likely
possibility, although intervention of carboxypeptidases cannot be
excluded. When examined in vivo, the stability of the
(6-7) analogue was also significantly increased. Its half-life in
the serum of injected mice was increased by >10 times compared with
GP33. The rapid disappearance of GP33 from serum (by renal clearance or
most probably as a result of proteolytic degradation), however, may be
balanced by the very fast loading of GP33 to MHC molecules from APC.
Ten minutes after peptide injection, GP33 (as well as the
(6-7)
peptide) was present on splenocytes. This rapid loading of exogenously
provided peptides suggests a direct binding to MHC molecules without internalization.
(6-7)-MHC complexes on APC was increased by a
factor of two. Several possibilities may account for this increased
stability of
(6-7)-MHC complexes. As shown in a stabilization assay
with RMA-S cells, the binding of
(6-7) and GP33 peptides to MHC
molecules was similar. Nevertheless, we cannot rule out the possibility that the affinity equilibrium constant of the parent peptide and the
(6-7) analogue to Db molecules are slightly different.
A second possibility is that reloading on the cell surface after
dissociation from the MHC groove is increased in the case of the
(6-7) analogue because this analogue probably also exhibits an
enhanced resistance to proteases present in the extracellular matrix.
Finally, it is possible that peptide-MHC complexes are internalized
after a few hours, and that because of increased proteolytic resistance
to cytoplasmic proteases, the
(6-7) analogue can be reloaded on MHC
molecules and represented at the cell surface.
(6-7) are loaded to MHC molecules
with a similar initial efficacy, when we assessed the immunogenic
activity of the
(6-7) analogue in antiviral protection experiments
(peptides injected in IFA), we did not find an improved T cell response
to the more stable peptide
(6-7). The pseudopeptide effectively
showed antiviral protection properties, which is a novel observation,
but these were not significantly different from those observed with
GP33. This result suggests that possibly because of the presence of oil
in the adjuvant, the advantage of increased proteolytic resistance and
prolonged in vivo persistence did not improve the apparent
biological activity of the analogue. Furthermore, once peptides are
bound to MHC molecules they are apparently protected from digestion
(37). It is known that injection of GP33 inoculated without adjuvant is
not able to protect mice from LCMV after challenge infection (Ref. 38
and Fig. 5B). An important observation shown in this study
is that three subcutaneous injections of the free analogue
(6-7) in
saline were able to reduce the virus titer in the spleen of immunized
mice. This result suggests that
(6-7) but not GP33 is able to prime
T cells in the absence of IFA. It remains to be analyzed whether this
result is attributable to the higher stability of the
(6-7)
analogue in the circulation or to the prolonged half-life of the
(6-7) peptide-MHC complexes. However, the reduction of virus titers obtained with
(6-7) in PBS was not as impressive as observed with
peptides in IFA, suggesting the need of an inflammatory process to
reach complete antiviral protection. Because our conclusions are of
immediate relevance to vaccination, it would be interesting to test the
presence and reactivity of memory CD8+ T cells generated
after pseudopeptide vaccination. Finally, to better understand the
possible mechanisms involved in the clearance of virus by
peptide-activated T cells, it will be important to examine the
immunological properties of modified peptide ligands containing
different types of amide bond isosteric replacements in different viral systems.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank S. Batsford and G. Guichard for comments on the manuscript, M. Rawiel for excellent technical assistance, and S. Denkler and T. Imhof for animal husbandry.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Deutsche Forschungsgemeinschaft Grant PI 295/3-1 and by grants from the Association pour la Recherche sur le Cancer and Agence Nationale de Recherche sur le SIDA.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a postdoctoral fellowship from Alexander Von Humboldt
Stiftung and from INSERM.
¶ Supported by a grant from La Ligue Nationale contre le Cancer.
To whom correspondence should be addressed: Institute for
Medical Microbiology and Hygiene, Department of Immunology, Hermann Herder Str. 11, D-79104 Freiburg, Germany. Tel.: 49-761-203-6521; Fax:
49-761-203-6577; E-mail: pircher{at}ukl.uni-freiburg.de.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MHC, major histocompatibility complex; APC, antigen-presenting cell; CTL, cytotoxic T lymphocyte; HPLC, high performance liquid chromatography; IFA, incomplete Freund's adjuvant; LCMV, lymphocytic choriomeningitis virus; PBS, phosphate-buffered saline; TCR, T cell receptor; Tg, transgenic.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|