From the Institut de Pharmacologie et de Biologie
Structurale, CNRS, 205 route de Narbonne, 31400 Toulouse, France and
the
Department of Neuropharmacology, Division of Virology,
Scripps Research Institute, La Jolla, California 92037
Received for publication, September 28, 2000, and in revised form, February 7, 2001
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ABSTRACT |
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Structural similarity (molecular mimicry) between
viral epitopes and self-peptides can lead to the induction of
autoaggressive CD4+ as well as CD8+ T
cell responses. Based on the flexibility of T cell
receptor/antigen/major histocompatibility complex recognition, it has
been proposed that a self-peptide could replace a viral epitope for T
cell recognition and therefore participate in pathophysiological
processes in which T cells are involved. To address this issue, we
used, as a molecular model of viral antigen, the
H-2Db-restricted immunodominant epitope
nucleoprotein (NP)-(396-404) (FQPQNGQFI) of lymphocytic
choriomeningitis virus (LCMV). We identified peptide sequences from
murine self-proteins that share structural and functional homology with
LCMV NP-(396-404) and that bound to H-2Db with high
affinity. One of these self-peptides, derived from tumor necrosis
factor receptor I (FGPSNWHFM, amino acids 302-310), maintained
LCMV-specific CD8+ T cells in an active state as observed
both in vitro in cytotoxic assays and in vivo
in a model of virus-induced autoimmune diabetes, the rat insulin
promoter-LCMV NP transgenic mouse. The natural occurrence and molecular
concentration at the surface of H-2b spleen cells of tumor
necrosis factor receptor I-(302-310) were determined by on-line
µ-high pressure liquid chromatography/mass spectrometry and
supported its biological relevance.
Molecular mimicry, a process in which potentially autoreactive T
cells are activated in the periphery by major histocompatibility complex (MHC)1-restricted
cross-reactive self-peptides, is suspected to be a possible mechanism
triggering pathological (versus normal) immunity, particularly virus-induced autoimmune disease (1). Although the
recognition of a viral antigenic peptide by the T cell receptor (TCR)
is a most specific process, structurally altered antigenic variants or
mimicry peptides, which can be unrelated in terms of primary sequence
to the viral antigen (2-4), can still be recognized by
CD4+ or CD8+ T cells (5, 6). The functional
properties of these peptides depend on their structure, and their
interaction with the TCR can lead to either full or partial T cell
activation or antagonism (5). Such cross-reactive peptides are thought
to play a role in pathophysiological situations such as T cell
selection (7, 8), anergy (9), viral escape (10, 11), and autoimmune disorders (12).
Although the development of autoimmune disease has been commonly
associated with CD4-bearing T cells (4), there are now both clinical
and experimental observations that strongly suggest that autoreactive
CD8+ T cells may also be involved (13, 14). However,
the molecular mechanisms by which autoreactive CD8+ T cells
are activated or maintained in a functional state remain mostly
unknown. In this study, we explored the possible role of MHC class
I-restricted self-peptides in these mechanisms because of their
potential importance in clinical disease. Our aim was (i) to identify
putative self-molecular mimics of a viral antigen, (ii) to characterize
their structural and functional properties, and (iii) to determine
their biological relevance. For this, we used the
H-2Db-restricted immunodominant epitope located in
nucleoprotein (NP)-(396-404) (FQPQNGQFI) of lymphocytic
choriomeningitis virus (LCMV), a model of choice for dissecting the
molecular or cellular mechanisms involved in an autoreactive
CD8+ T cell response. Indeed, the
H-2Db-restricted CD8+ T cell response mounted
against the LCMV NP is directed toward the immunodominant epitope
sequence NP-(396-404) (15-17). The LCMV-infected mouse
typically undergoes activation and massive expansion of CD8+ T cells, which consistently stay at a high level
throughout the animal's life span, even in the absence of detectable
virus and/or viral antigen (18-20). These cytotoxic T cells (CTLs)
represent a potential source of autoreactivity, particularly if their
cytolytic function remains intact. The H-2b transgenic
mice, which express LCMV NP under the control of the rat insulin
promoter (RIP), represent an in vivo model of autoimmune disease (21, 22) in which LCMV NP-(396-404) can be the target of
autoreactive CTLs. After LCMV infection or adoptive transfer of
anti-LCMV CTLs, these RIP-LCMV NP mice develop
insulin-dependent diabetes mellitus (IDDM), a virus-induced
autoimmune disease in which both CD4+ and CD8+
T cells are involved (23). In these mice, the destruction of Here, we first identified a set of six nonameric sequences from
endogenous proteins sharing structural and functional homology with
LCMV NP-(396-404). Five of these endogenous peptides bound with high
affinity to H-2Db and generally acted as antagonists of
lysis by LCMV-specific CTLs. In the presence of H-2b cells
pulsed with three of these peptides, LCMV-specific CTLs were maintained
in long-term culture. We then selected one of these peptides, tumor
necrosis factor receptor I (TNFR)-(302-310), which activated LCMV
NP-specific CTLs, allowing them to kill LCMV-infected cells in a
peptide-specific, MHC-restricted manner. Adoptive transfer of
TNFR-(302-310)-activated CTLs into RIP-LCMV NP × RIP-B7.1
transgenic mice provoked a specific destruction of Cell Lines, Mice, and CTLs--
The T2 human mutant cell line
transfected with H-2Db (T2-Db) (26) was used in
binding experiments. The murine H-2b cell lines MC57 and
RMA and the H-2d cell line BALB/cl7 were used as
target cells in CTL assays. Cells were grown in RPMI 1640 medium (MC57,
RMA, and BALB/cl7) or Iscove's modified Dulbecco's medium
(T2-Db) containing 8% bovine serum,
L-glutamine, and antibiotics. Geneticin (400 µg/ml) was
added to Iscove's modified Dulbecco's medium to maintain selection of
positively transfected T2-Db cells. C57BL/6
(H-2b) and BALB/c (H-2d) mice were obtained
from the breeding colony at the Scripps Research Institute. CTL clone
NP18, specific for the H-2Db-restricted LCMV epitope
NP-(396-404) (27), was restimulated weekly with LCMV-infected MC57
cells and spleen cells from C57BL/6 mice. Polyclonal CTLs were
generated by cultivating naive spleen cells from C57BL/6 mice with 1 µM LCMV NP-(396-404) without interleukin-2 for 10 days
and then restimulated weekly using LCMV NP-(396-404)-coated RMA cells
and spleen cells from C57BL/6 mice in the presence of interleukin-2 (30 units/ml). CTLs used in long-term culture studies or adoptive transfer
experiments were generated from C57BL/6 and control BALB/c mice
infected with 105 plaque-forming units of the LCMV
Arm strain intraperitoneally. 45-60 days later, a single
suspension of spleen cells was cultured with or without peptide-pulsed
(1 µg/ml) or LCMV-infected macrophages.
MHC Binding Studies--
Peptide binding to H-2Db
was determined in a competition assay as previously described (28).
T2-Db cells (1 × 105 cells/well) were
incubated with fluorescein isothiocyanate-labeled KAIENAEAL (100 nM) for 75 min at 37 °C in the
presence of proteases inhibitors. Total binding and nonspecific binding
were measured in the absence or presence of 1 mM unlabeled
peptide, respectively. Cells were washed with 1% bovine serum albumin
in phosphate-buffered saline and fixed in 1% paraformaldehyde, and the
fluorescence (mean fluorescence intensity (mfi)) was analyzed by
fluorescence-activated cell sorting (Becton-Dickinson). Assays were
performed with increasing concentrations
(10 Peptide Search in Data Bases--
An algorithm was used to
search for murine nonameric peptides sharing functional homology with
LCMV NP-(396-404) in the Swiss Protein Database. Criteria (illustrated
in Fig. 1) were as follows: at P1, all 20 amino acids except Leu, Ile,
Met, and Val; at P2, Ala, Asn, Cys, Gln, Gly, and Ser; at P3, Leu, Met,
Ile, Val, Pro, and Ala; at P4, all amino acids; at P5, Asn; at P6, all
amino acids; at P7, all amino acids; at P8, Phe; and at P9, Leu, Ile, and Met. Restrictions at P1, P2, P3, P5, and P9 were imposed according to published studies (29-31).
In Vitro Cytotoxic Assays--
LCMV-infected or peptide-pulsed
uninfected target cells were incubated for 1 h at 37 °C with
51Cr and washed, and LCMV-specific CTLs were added at the
indicated effector/target ratio. Target and effector cells were
incubated at 37 °C in a final volume of 200 µl. After a 5-h
incubation period, fractions (100 µl) were removed and counted for
51Cr activity. The percent specific lysis was calculated as
100 × ((cpm(experimental release) IFN- Proliferation Assay--
CTLs (1 × 104) were
mixed with RMA cells (5 × 104) previously
Adoptive Transfer and in Vivo Immunopathology
Assay--
H-2b or H-2d double RIP-LCMV
NP × RIP-B7.1 transgenic mice expressing LCMV NP × B7.1 in Extraction of Endogenously Presented Peptides from Spleen
Cells--
Peptides were acid-extracted from the cell surface as
previously described (32). Briefly, splenocytes (1-2 × 109 cells) were washed three times with phosphate-buffered
saline and then resuspended in 0.131 M citric acid and
0.066 M Na2HPO4 at pH 3.0 for 2 min. The eluted material was desalted on Waters Sep-Pak column
according to the manufacturer's instructions, vacuum-concentrated, and
centrifuged in 1% trifluoroacetic acid on a Centricon 3 (3-kDa cutoff;
Amicon, Inc.) at 3800 × g for 90 min at 4 °C. The
filtered material was vacuum-concentrated and resuspended in 100 µl
of 0.08% trifluoroacetic acid. Peptides were separated on a
reversed-phase C18 column (Aquapore, 7 µm, 2.1 × 100 mm; Brownlee) using a Waters 600S controller system. Samples (25 µl) were injected and separated using a system gradient of 5-36%
solvent B for a 60-min period at a flow rate of 200 µl/min. Solvent A
was 0.08% trifluoroacetic acid in H2O, and solvent B was
0.08% trifluoroacetic acid in CH3CN. Fractions (200 µl)
were collected, lyophilized, and stored at Structural Identification of Naturally Occurring Peptides by
On-line µ-HPLC/ESI-MS--
The collected HPLC fractions were
injected on a reversed-phase C18 microcolumn
(PepMapTM, 3 µm, 0.3 × 150 mm; LC Packings).
Elution was performed with a gradient of 5-55% solvent B in 25 min.
Solvent A was 0.05% acetic acid in 95:5 (v/v)
H2O/CH3CN, and solvent B was 0.05% acetic acid in 20:80 (v/v) H2O/CH3CN. The flow rate was set
at 400 µl/min and split to 4 µl/min before the column. MS data were
recorded on a Finnigan MAT TSQ700 triple quadrupole mass spectrometer
equipped with an electrospray source either by scanning the range of
masses corresponding to m/z values between 300 and 1800 every 3 s or by using the "single ion monitoring" mode, where
the quadrupole is set to transmit only one particular m/z
value, corresponding to an ion of interest, centered in a
1-m/z unit window. Low energy collision-activated
dissociation MS/MS experiments were conducted in the "selected
reaction monitoring" mode, where the first quadrupole is set to
transmit the precursor ion of interest and the third quadrupole is
scanned over a 10-m/z unit mass range centered on the
m/z value of the fragment ion of interest. Argon was used as
the collision gas at a collision pressure of 1.2 millitorr. The
collision energy was 28 eV in the laboratory frame of reference.
Molecular Modeling of H-2Db/Peptide
Interactions--
The H2-Db structure was obtained from
crystallographic data of H-2Db with influenza virus peptide
NP-(366-374) (33). The LCMV NP-(396-404) and TNFR-(302-310) nonamers
were introduced manually using the program O. Side chain conformation
was based on the most probable rotamer and the probable hydrogen bonds.
After transfer of H-2Db data to Insight II (Biosym
Technologies), minimization was done using the VA09A (1000 iterations
without constraints) algorithm.
Structural and Functional Anatomy of the LCMV NP-(396-404)
Epitope--
We first identified the residues of LCMV NP-(396-404)
involved in MHC binding and those serving as TCR contacts. Binding
assays of monoalanine-substituted analogs (Table
I) confirmed Asn400 at P5 and
Ile404 at the C terminus as H-2Db anchors.
Pro398 at P3 contributed significantly to MHC binding,
likely by pointing down to the MHC-binding groove (33) and/or, as a
proline residue, by inducing structural constraints to the peptide
backbone (34). The impact of alanine substitution on the ability of
NP-(396-404) to sensitize target cells to lysis by LCMV-specific CTLs
was then assessed. Results obtained with the NP-(396-404)-specific CTL clone NP18 (35) are shown in Table I. The strongest effect was observed
at position 8, where substitution of Ala for Phe yielded a peptide
totally unable to sensitize target cells even at the highest
concentration tested, indicating that Phe403 is the
critical (main) TCR contact. CTL sensitization properties were also
profoundly altered, but not abolished, after substitution of
Phe396 at P1 and Gln399 at P4, indicating that
these two residues also play a role, although of lesser importance, as
TCR (auxiliary) contacts. Substitution of residues at positions 2 (Gln), 6 (Gly), 7 (Gln), and 9 (Phe) had little effect. The effects
observed at positions 3 and 5 were expected as a consequence of the
weak MHC-binding properties of the analogs. Similar results were
obtained with polyclonal populations of CTLs obtained from C57BL/6 mice
either infected with LCMV Arm or immunized with synthetic
NP-(396-404) (data not shown). These three main (Phe403)
and auxiliary (Phe396 and Gln399) TCR contacts
identified in the NP-(396-404) sequence undoubtedly represent the
overall CTL response against this antigen. A summary of MHC binding and
TCR interactions is illustrated in Fig.
1A.
Search for Murine Self-peptides Sharing Minimal Functional Homology
with LCMV NP-(396-404)--
We next screened the Swiss Protein
Database for murine nonameric self-peptides fulfilling the following
requirements (Fig. 1B). The two H-2Db anchors
must be present at P5 (Asn) and P9 (Met, Leu, or Ile) (29) as well as
the main CTL contact at P8 (Phe) of NP-(396-404). Additional criteria
were imposed at P1, P2, and P3, for which some residues affect peptide
presentation by H-2Db (30, 31). Finally, no restriction was
applied to P4, P6, and P7, at which structural modification has no or
little impact on NP binding. About 30 peptides were extracted from the
registered murine proteins, with a high proportion of redundant
sequences (i.e. different members of the same protein family
registered under different code numbers). From them, we selected the
only six sequences from non-redundant proteins (Table
II). In addition to the two main MHC
anchors and the main TCR contact (minimal functional homology), some of
these peptides presented a higher sequence homology by sharing one or
two other residues with LCMV NP-(396-404). The six murine
self-peptides (mimicry peptides) were then tested for binding to
H-2Db. As shown in Table II, five of them bound efficiently
to H-2Db with affinities comparable to (lysosomal
acid phosphatase-(177-185) and lactate dehydrogenase-(282-290)),
higher than (protein kinase C-(351-359) and TNFR-(302-310)), or lower
than (N-cadherin-(569-577)) that of NP-(396-404). Only one peptide,
IgVH-(91-99) (Ig V region heavy
chain), was a weak H-2Db binder and, for this reason, was
not studied further.
Recognition of the Murine Self-peptides by LCMV
NP-(396-404)-specific CTLs--
The capacity of the five mimicry
peptides to activate such functions of LCMV NP-specific CTLs as
cytotoxicity, IFN- The Murine Self-peptides Homologous to LCMV NP-(396-404) Are
Antagonists of the NP-specific TCR--
Since structural modification
can alter TCR recognition by provoking partial agonism or antagonism
(36-39), we next determined if the self-peptides could behave as
antagonists. Fig. 2D depicts the reduced lytic function of
the NP-specific CTLs by the murine self-peptides with minimal homology
to NP-(396-404). Lysis of labeled target cells coated with suboptimal
concentrations of NP-(396-404) was inhibited in the presence of
increasing concentrations of synthetic self-peptides, i.e.
50-90% inhibition was reached at the highest concentration tested
(100 µM). In negative control experiments, no antagonism
was detected with the two other H-2Db-restricted LCMV
immunodominant epitopes: glycoprotein-(276-286), which binds to
H-2Db with a high affinity, similar to that of
NP-(396-404) (15) (Fig. 2), and glycoprotein-(33-41) (data not shown).
Long-term Maintenance of LCMV-specific CTLs by Mimicry
Peptides--
To determine whether mimicry peptides could maintain
anti-LCMV CTLs in long-term culture, we used single suspensions of
spleen cells from C57BL/6 mice 45-60 days after LCMV infection. Such splenocytes contain numerous anti-LCMV and particularly
anti-NP-(396-404) memory CTLs (17). These CTLs were cultured
with macrophages coated with peptide, infected with LCMV, or left
untreated. The three peptides selected for this study,
N-cadherin-(569-577), protein kinase C-(351-359), and
TNFR-(302-310), shared three, four, and five functional residues with
LCMV NP-(396-404), respectively (see Table II). As summarized in Table
III, in the absence of stimulatory signals, T cells died in culture within 2 weeks, whereas stimulation by
cells infected with LCMV or pulsed with the synthetic LCMV NP-(396-404) peptide routinely resulted in a long-term maintenance of
anti-LCMV CTLs. The three mimicry peptides tested allowed the anti-LCMV
CTLs to grow in culture in the long-term range. In contrast, CTLs died
within 2 weeks when cultured in the presence of unmatched MHC
(H-2d) macrophages pulsed with the
H-2Ld-restricted LCMV NP-(118-126) immunodominant
epitope.
In Vitro and in Vivo Activities of Long-term Cultured CTLs
Stimulated with TNFR-(302-310)--
We next determined whether
antiviral CTLs maintained in long-term culture in the presence of a
mimicry peptide retained functional MHC-restricted activity. We used
the self-peptide TNFR-(302-310) for these studies. As presented in
Table IV, CTLs stimulated with TNFR-(302-310) lysed LCMV-infected H-2b targets. However,
the efficiency was lower than that observed with CTLs stimulated with
LCMV or NP-(396-404). In control experiments, H-2b targets
pulsed with the synthetic NP-(396-404) or TNFR-(302-310) peptide were
also lysed by CTLs, whereas H-2d targets coated with the
H-2Ld-restricted epitope NP-(118-126) were not
lysed (data not shown).
We then determined whether the TNFR-(302-310)-stimulated CTLs had
preserved cytotoxic activity in vivo. For this purpose, such
CTLs were adoptively transferred into double RIP-LCMV NP × RIP-B7.1 transgenic mice. In such mice, either LCMV infection or
adoptive transfer of anti-NP-(396-404) CTLs causes CTL infiltration into the islets of Langerhans, insulitis, and IDDM as measured by the
elevation of blood glucose and reduction of pancreatic insulin (21, 22,
40). As shown in Table V, insulitis and elevated blood glucose levels indicating IDDM were observed in recipients of adoptively transferred LCMV-specific CTLs previously co-cultured with TNFR-(302-310) for >2 months. Immunohistochemical analysis confirmed CTL infiltration into the pancreases of these mice
(data not shown). Specificity was demonstrated when H-2d
transgenic mice failed to develop insulitis or IDDM after adoptive transfer of H-2b-restricted CTLs primed by
TNFR-(302-310)-coated or LCMV-infected macrophages.
The TNFR-(302-310) Peptide Is Naturally Presented at the Surface
of H-2b Spleen Cells--
The final and important step was
to document that such a self-peptide with the ability to maintain
functional virus-specific CTLs was present at the cell surface. To do
so, peptides were extracted from the surface of splenocytes, separated
by HPLC, collected into fractions of interest, and then analyzed by
on-line µ-HPLC/ESI-MS. Because the TNFR-(302-310) peptide contained
a methionine residue, its chemical modification was expected during the
extraction and analysis process. Indeed, as a control, the synthetic
TNFR-(302-310) peptide eluted in two distinct peaks, fractions 52 and
58 (Fig. 3, STEP-1,
F52 and F58). As assessed by MS, fraction 58 corresponded to the unmodified form of the synthetic peptide ((M + 2H)2+, m/z 561.9), and fraction 52 corresponded
to the chemically modified form bearing an oxidized C-terminal
methionine ((M + 2H)2+, m/z 569.9; as shown in
Fig. 3, STEP-1, panel a, inset). When analyzed at different concentrations (10-1000-fold dilutions), we
noted that the susceptibility of TNFR-(302-310) to oxidation both
under sample storage conditions and during analysis increased with
peptide dilution (data not shown). Peptides extracted from C57BL/6
(H-2b) and control BALB/cl7 (H-2d)
spleen cells were then separated, and their respective fractions 52 were collected and analyzed by on-line µ-HPLC/ESI-MS (Fig. 3,
STEP-2). An ion whose mass and retention time were identical to those of the oxidized form of the synthetic TNFR-(302-310) peptide
was clearly detected in fraction 52 of H-2b (but not
H-2d) spleen extracts. Identification of this natural form
of TNFR-(302-310) was unambiguously confirmed by (i) overloading
experiments, which gave a perfectly superimposable coelution of the
natural product and the overloaded synthetic oxidized form; and (ii)
MS/MS experiments, which produced a fragment ion specific for the
oxidized TNFR-(302-310) peptide upon collision, as detected by
selected reaction monitoring (Fig. 3, STEP-2, right
panels). Based on the measured ionic current intensity, the amount
of the TNFR-(302-310) peptide extracted from 108
H-2b spleen cells was ~120 fmol. Given the yield of the
extraction procedure (10-15%), the amount of TNFR-(302-310) detected
on the cell surface was estimated at 1 pmol/108 cells,
which represents an approximate number of 7200 molecules/cell.
Structural similarity (molecular mimicry) between viral epitopes
and self-peptides can lead to the induction of MHC class II-restricted
autoaggressive CD4+ T cells (4) as well as MHC class I
CD8+ T cell responses (13, 14, 41, 42). Both clinical and experimental evidence continues to accumulate that infectious agents
encoding proteins that cross-react with host self-proteins can play a
role in molecular mimicry (43, 44).
Data base search for peptides sharing structural homology with a given
antigen may be successfully employed to identify potential cross-reactive endogenous ligands (1, 4, 42). Using this strategy, we
were able to identify at least five murine self-peptides with
predictable high H-2Db-binding affinity that share with the
LCMV NP-(396-404) antigen its main TCR contact residue (Phe at P8).
Among these five peptides, three also shared one of the two auxiliary
TCR contact residues, either Phe at P1 (protein kinase C-(351-359) and
TNFR-(302-310)) or Gln at P4 (lysosomal acid phosphatase-(177-185)).
In addition, the TNFR-(302-310) peptide had a third residue in common
with LCMV NP-(396-404) (Pro at P3), resulting in a marked functional homology between the viral epitope and the self-peptide (three out of
three MHC-binding residues and two out of three TCR contact residues).
Yet, we failed to find a murine self-peptide that bore the three TCR
contacts of the viral antigen. One may consider that the number of
peptides identified by our method was unexpectedly small. However, the
small set of self-peptides we found (<10) resembled that of other
Db-restricted (7) or Kb-bound (8) peptides
implicated in T cell-positive selection and identified using different
approaches. Also, a search for mimicry peptides able to interact with
MHC class II-restricted autoreactive T cell clones specific for the
myelin basic protein produced a similarly small group (4). These
previous results validate our approach, which is further strengthened
by the identification of one of these peptides (TNFR-(302-310)) in the
material eluted from the surfaces of H-2b cells.
By which molecular mechanism(s) can the naturally occurring
TNFR-(302-310) peptide and the other self-peptides maintain
potentially autoreactive CTLs in a functional state? With respect to
the NP-specific TCR, TNFR-(302-310) and the other self-peptides
identified here behaved as typical altered peptide ligands (45), with
implications at both functional and structural levels. At the
functional level, despite the presence in their sequences of the main
TCR contact and (for some of them) of an additional auxiliary TCR
contact, none of the mimicry self-peptides identified here were able to stimulate antiviral CTLs in agonist assays. Nevertheless, they behaved
as antagonists, indicating that they still interacted with the TCR and
suggesting that their affinity for the NP-specific TCR was relatively
low (7). Low affinity recognition of self-MHC promotes thymocyte
survival, and in this way, the self-peptides act as low affinity
molecular mimics (46). The TCR antagonism we observed may result from
impaired TCR triggering. To dissect these TCR-triggering mechanisms
further would require exploration of the self-peptides' ability to
induce, or not, events such as TCR internalization, TCR dimerization,
and phosphorylation of the kinases involved in signal transduction
(47). At the structural level, our results support previous studies
showing that degenerate recognition of the TCR is not limited to singly
substituted variants of a given antigen, but also applies to peptides
of originally unrelated sequences (3, 7, 8, 48, 49). Molecular modeling
(Fig. 4) revealed that the backbones of
LCMV NP-(396-404) and the self-peptide bound to H-2Db
could adopt superimposable conformations. The side chain of Phe at P8
was oriented similarly in the different peptides, a structural feature
essential for preserving the function of this residue as the main TCR
contact. In contrast, substitution of the viral residue Gly (with no
side chain) at P6 for a bulkier self-residue whose side chain points
out of the MHC groove clearly created a structural alteration of
the peptide-MHC complex surface susceptible to modify
(inhibit) the local interactions between the TCR and the MHC
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells
and ensuing IDDM have been attributed to MHC class I-restricted CD8+ T cells (22, 24, 25). Furthermore, the finding that
IDDM does not develop in MHC class I-deficient mice or CD8-depleted mice (24) provides additional support for the involvement of CD8+ T cells and, consequently, of MHC class I-restricted
self-peptides.
cells of the
islets of Langerhans and caused IDDM. Using on-line µ-high pressure
liquid chromatography (HPLC)/electrospray ionization mass spectrometry (ESI-MS), we demonstrated the presence of TNFR-(302-310) at the surface of H-2b spleen cells, a finding supporting its
biological relevance.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10 to 10
5
M) of unlabeled competitors. Specific binding to
H-2Db was defined as the difference between total and
nonspecific binding. The percentage inhibition of binding was
calculated as 100 × (1
(mfi in presence of
competitor
(mfi(nonspecific binding)/mfi(specific binding)))).
The 50% inhibiting concentration (IC50) corresponds to a
peptide concentration inhibiting half the maximal specific binding of
fluorescein isothiocyanate-labeled KAIENAEAL to H-2Db.
cpm(spontaneous
release))/(cpm(total release)
cpm(spontaneous release))). Total
release and spontaneous release were determined by incubating the
labeled cells with 1% Nonidet P-40 and culture medium, respectively.
In all experiments, samples were run in triplicate, and the mean values
are given. Antagonism was assayed in a 5-h 51Cr release
assay as described above, except that RMA cells were prepulsed with a
suboptimal concentration of LCMV NP-(396-404) (giving 30-40% lysis)
during 51Cr labeling, extensively washed, and incubated
with a 1, 10, or 100 µM concentration of the indicated
peptides for 30 min at 37 °C before CTLs were added at an
effector/target ratio of 5:1.
Production--
RMA cells (3 × 103
cells/well) were incubated for 1 h at 37 °C in the presence of
the indicated peptides (1 × 10
10 to
1 × 10
5 M). CTLs (15 × 103 cells/well) were then added and incubated with
target cells for 24 h at 37 °C. The IFN-
present in the
supernatant was measured by enzyme-linked immunosorbent assay using
R4-6A2 and biotinylated XMG1.2 as the pair of antibodies (a kind gift
of Dr. J.-C. Guery). Horseradish peroxidase-conjugated streptavidin
(Amersham Pharmacia Biotech) was used to detect biotinylated
XMG1.2, followed by incubation with
o-phenylenediamine hydrochloride (Sigma). The color was red at 490 nm using an enzyme-linked immunosorbent assay reader (Titertek Multiskan Plus MKII, EFLAB, Finland) and normalized to the
values obtained for a standard curve of recombinant murine IFN-
(Sigma).
-irradiated (10,000 rads) and pulsed with peptide (1 µM). Cells were incubated for 72 h at 37 °C and
with 1 µCi of [3H]thymidine during the last 16 h.
The level of [3H]thymidine incorporation was then
determined by scintillation counting.
cells of islets of Langerhans were adoptively
transferred with 2 × 107 long-term cultured T
cells maintained as described above and inoculated intraperitoneally.
Blood glucose levels were monitored, and pancreatic tissues were
studied by immunohistochemistry for evidence of CTL infiltration as
previously described (22, 24, 40).
80 °C until analysis by
on-line µ-HPLC/ESI-MS.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
H-2Db binding and CTL recognition of the LCMV NP-(396-404)
epitope and monoalanine-substituted analogs
10 to 10
5 M)
were tested in a MHC class I competition assay on T2-Db cells
as described previously (28). IC50 values represent the peptide
concentrations inhibiting half the maximal binding of the labeled
peptide. Values are the means ± S.E. of three independent
experiments. For CTL recognition, peptides (10
12 to
10
6 M) were tested in a classical CTL assay by
measuring the lysis of peptide-coated MC57 target cells by NP-specific
CTL clone NP18 (effector/target ratio of 5:1). EC50 values
represent the peptide concentrations inducing half of the maximal lysis
effect. Values from one experiment are shown and are representative of
several independent experiments. Residues implicated in either MHC
binding or TCR recognition are shown in boldface italic type.
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Fig. 1.
Screening strategy for murine self-peptides
that have minimal functional homology to LCMV NP-(396-404).
A, anatomy of the viral epitope. Residues implicated in MHC
binding or in TCR interactions were identified using the monoalanine
scanning approach. The main and auxiliary interactions are shown as
black and stippled arrows, respectively.
B, search criteria for nonameric self-peptide sequences
sharing minimal functional homology with LCMV NP-(396-404)
(FQPQNGQFI). We searched the Swiss Protein Database by (i) fixing the
required functional amino acids at P5, P8, and P9; (ii) imposing or
excluding some amino acids known to play a positive (at P2 and P3) or
negative (at P1) role in H-2Db binding (31); and (iii)
allowing any of the 20 natural amino acids at P4, P6, and P7.
Self-peptides from murine proteins with minimal functional homology to
LCMV NP-(396-404)
production, proliferation, and Ca2+
mobilization was then evaluated. As shown in Fig.
2A, no significant lysis of
target cells was observed in the presence of the self-peptides. LCMV
NP-(396-404) induced a strong IFN-
production (Fig. 2B) and T cell proliferation (Fig. 2C), as expected. In contrast, none of
the self-peptides incited either IFN-
production or measurable T
cell proliferation in vitro. Similar results were obtained
when Ca2+ mobilization was analyzed (data not shown).
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Fig. 2.
Functional properties of murine self-peptides
upon reacting with H-2Db class I-restricted LCMV
NP-(396-404)-specific CTLs. A, sensitization
of target cells to lysis by CTL clone NP18 (panel a) or
polyclonal CTLs (panel b) specific for LCMV NP-(396-404)
was analyzed in a 51Cr release assay as described under
"Experimental Procedures." B, IFN- production.
Enzyme-linked immunosorbent assays were carried out as described for
the cytotoxicity assays, except that supernatants were collected after
24 h and tested for IFN-
content. Results are expressed as
international units/ml and are representative of two independent
experiments. C, proliferation. T cells (1 × 104) were mixed with
-irradiated, peptide-pulsed RMA
cells (5 × 104) and incubated for 72 h at
37 °C. [3H]Thymidine was added during the last 16 h, and incorporation was determined by scintillation counting.
D, T cell antagonism. The antagonist properties of the
self-peptides were assessed in cytotoxicity assays. RMA cells were
loaded with a suboptimal concentration (giving <50% maximal lysis) of
LCMV NP-(396-404); washed; and incubated with a 1, 10, or 100 µM concentration of the indicated peptide before addition
of CTLs. Results are from one representative experiment among three.
NCad, N-cadherin; LAP, lysosomal acid
phosphatase; LDH, lactate dehydrogenase; PKC,
protein kinase C
; GP, glycoprotein.
Fate of LCMV-specific H-2b CTLs as culture conditions vary
.
In vitro cytotoxic activity of long-term cultured LCMV-specific CTLs
In vivo activity of LCMV-specific CTLs after adoptive transfer into
H-2b RIP-LCMV NP × B7.1 transgenic mice
cells of islets of Langerhans using the RIP were used. Mice
were adoptively transferred with 2 × 107 long-term
cultured CTLs activated as described in the legend to Table III and
inoculated intraperitoneally. Blood glucose (BG) levels were monitored,
and CTL infiltration of pancreatic tissues was studied as described
(22, 24, 40).
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Fig. 3.
On-line µ-HPLC/ESI-MS
identifies the naturally occurring self-peptide TNFR-(302-310) on the
surfaces of H-2b spleen cells. In the first
step (STEP-1), acid-eluted peptides from C57BL/6
(H-2b) or Dba/2 (H-2d) splenocytes or a mock
extract of synthetic TNFR-(302-310) were fractionated by HPLC as
described under "Experimental Procedures." The oxidized and
unmodified forms of TNFR-(302-310) eluted in fractions 52 (F52) and 58 (F58), respectively. The mass
spectrum of fraction 52 is shown in the inset in panel
a (m/z 569.9 corresponds to the (M + 2H)2+ ion of the oxidized form). In the second step
(STEP-2), the respective fractions 52 were collected and
further analyzed by on-line µ-HPLC/ESI-MS (left panels)
using the single ion monitoring detection mode at m/z 569.9. Plots show the ion current from aliquots corresponding to 1 × 108 cells. Low energy collision-activated dissociation
MS/MS experiments were conducted in the selected reaction monitoring
mode (right panels), where the first quadrupole is set to
transmit the precursor ion of interest (m/z 569.9),
corresponding to FGPSNWHFM, and the third quadrupole is scanned over
the 10-m/z unit mass range centered on the m/z
value of the fragment ion of interest (m/z 468.0),
corresponding to PSNWHFM. a.u., arbitrary units.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chains. Indeed, alterations of the peptide structure not directly
affecting TCR contacts may influence, locally or at distance, the
determinants and/or the antigenic surface of the peptide-MHC complex
interacting with the TCR (50-53). Furthermore, the presence of peptide
side chains that sterically hinder the TCR interaction with the MHC
helices results in lowered TCR affinity (54).
View larger version (41K):
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Fig. 4.
Side view of the superimposed structures of
LCMV NP-(396-404) and TNFR-(302-310) in the H-2Db-binding
groove. The 1- and
2-MHC
domains are shown as gray ribbons. The peptides are shown as
blue (LCMV NP-(396-404), FQPQNGQFI) or green
(TNFR-(302-310), FGPSNWHFM) sticks. The model shows the
perfect superimposition of the two peptide backbones and of the side
chains of the shared residues Phe1, Pro3,
Asn5, and Phe8. The model also illustrates the
steric hindrance of the bulky side chain of Trp6 of
TNFR-(302-310), which points out of H2-Db and likely
results in altered TCR/peptide/MHC interactions.
What pathophysiological role can a self-peptide like TNFR-(302-310)
play? The identification of TNFR-(302-310) as a self-peptide naturally
presented by MHC class I molecules lends support for its biological
relevance. The TNFR-(302-310) peptide originates from the
TNFR-I protein, a cell-surface receptor for tumor necrosis factor- (55). TNFR-I is highly expressed in the thymus, spleen, and
liver (56). In accord, we found that TNFR-(302-310) was naturally
present on the surfaces of H-2b spleen cells at 1 pmol/108 cells, a level comparable to that of other
self-peptides on the surfaces of thymic epithelial cells and implicated
in positive selection of CTLs (7). Evidence that TNFR-(302-310) has a
role in vivo in shaping the peripheral T cell repertoire, as
its natural presence on spleens cells might suggest, would require
further experimentation.
In conclusion, the work presented here addresses some fundamental
issues regarding the relationship between self- and foreign antigenic
peptides in the immune response. In particular, the central point made
is that self-peptides such as TNFR-(302-310) and others identified in
this study can allow antiviral, potentially autoreactive T cells to
keep their cytolytic function in the absence of viral antigen over a
period of months. Dissecting the mechanisms by which potentially
autoreactive CD8+ T cells are functionally maintained or
become activated should provide not only a better fundamental knowledge
of pathological autoimmunity, but also new therapeutic concepts for
combating it (57). In this study, we focused on MHC class I
self-peptides and hypothesized on their possible role in these
mechanisms. Because of the potential importance of these self-peptides
in clinical disease, the findings presented here may have important
consequences, particularly in terms of therapeutic application, by
helping to design peptidic or (better) non-peptidic molecular mimics to
target and/or inhibit potentially autoreactive T cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Honoré Mazarguil for peptide synthesis, Bernard Masri for preparation of peptide extracts, and Monique Erard for molecular modeling.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from CNRS; Association pour la Recherche sur le Cancer Contract 5485, Région Midi-Pyrénées; National Institutes of Health Grants AI41439, AI09484 (to M. B. A. O.), and AI44451 (to M. G. v. H.); and a Juvenile Diabetes International Foundation career development award (to M. G. v. H.). This is Publication 13360-NP from the Department of Neuropharmacology, Scripps Research Institute (La Jolla, CA).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.
§ These authors contributed equally to this work.
¶ Present address: INSERM U395, CHU Purpan, 31059 Toulouse Cedex, France.
** To whom correspondence should be addressed. Tel.: 33-561-175-530; Fax: 33-561-175-532; E-mail: gairin@ipbs.fr.
Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M008864200
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ABBREVIATIONS |
---|
The abbreviations used are:
MHC, major
histocompatibility complex;
TCR, T cell receptor;
NP, nucleoprotein;
LCMV, lymphocytic choriomeningitis virus;
CTL, cytotoxic T lymphocyte;
RIP, rat insulin promoter;
IDDM, insulin-dependent diabetes
mellitus;
TNFR, tumor necrosis factor receptor;
HPLC, high pressure
liquid chromatography;
ESI-MS, electrospray ionization mass
spectrometry;
IFN-, interferon-
.
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