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Centro de Biología Molecular Severo Ochoa (C.S.I.C.-U.A.M.), Universidad Autónoma de Madrid, Facultad de Ciencias, Madrid, Spain; and
Department of Pharmacy, ETH, Zürich, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The molecular basis for alloreactivity is poorly understood. MHC proteins constitutively present at the cell surface a large peptide repertoire, and the clonal diversity typical of alloreactive T cell responses is probably due to involvement of many MHC-bound peptides in allorecognition (1, 2, 3, 4). However, alloreactive T cells may exhibit various degrees of peptide specificity (2), and a few might be peptide independent or recognize motifs common to many peptides (5, 6, 7).
Various major issues remain to be solved. One is the nature of the peptides involved in allorecognition. To our knowledge, only a natural HLA ligand recognized by a human alloreactive CTL has been reported (8), and very few others have been identified in the mouse system (9, 10, 11). Therefore, the requirements for HLA-bound peptides to be immunogenic in alloreactive responses, such as affinity for MHC, expression level, and putative immunodominant character, have been insufficiently addressed. For instance, some of the peptides identified as allospecific epitopes were abundant and immunodominant (12, 13), but others were expressed at very low levels (8, 11).
A second issue is the molecular basis for the cross-reactions of alloreactive CTL. Self-restricted T cells can cross-react with peptides showing minimal homology to the nominal epitope (14, 15, 16). In one instance, an alloreactive CTL recognized two unrelated peptides in the context of two different allo-MHC molecules (17), and a third peptide in self-restricted fashion (18). In addition, a peptide isolated from a library that was recognized by an alloreactive T cell clone was different from the natural ligand recognized by that CTL (11, 19). In previous studies, alloreactive T cell clones raised against HLA-B27 (B*2705) showed limited cross-reaction with non-B27 Ags, but cross-reacted frequently with other HLA-B27 subtypes (20). Whether these cross-reactions were due to recognition of the same or different peptides in the various contexts was not determined.
A third issue is the role of the structural features of the MHC molecule in allospecificity. Although probably only a limited subset of alloreactive T cells might be peptide independent (4, 7), x-ray diffraction studies show that about 75% of the surface area contacted by the TCR corresponds to the MHC molecule (21, 22). However, the actual contribution of MHC residues, relative to bound peptide, to the alloantigenic determinants, and to the affinity for allospecific TCR remains to be established.
In this work, we report the identification of a natural peptide ligand of HLA-B27 that is recognized by a human alloreactive CTL clone raised against B*2705. We further establish the relationship between recognition of other HLA-B27 subtypes by this CTL clone and presentation of the peptide in the various contexts, and demonstrate the contribution of polymorphic HLA-B27 residues to the allospecific epitope. The same CTL clone distinguished between its peptide epitope and another closely related natural HLA-B27 ligand. The structure of the allospecific epitope and the molecular basis for this discrimination were analyzed by molecular modeling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-B*2705 alloreactive CTL 27S69 clone and its culture conditions have been described (20). It was raised from donor SR (HLA-A3, 29; B7, 44; DR2, 7) by in vitro stimulation with LCL4 R69 (HLA-A3, 24; B*2705, 7; DR3, 5). Besides B*2705, CTL 27S69 recognized B*2702, B*2703, and HLA-B61, but not B*2701, B*2704, or B*2706, as established with HLA-typed LCL.
HLA-B27 transfectant cell lines
Hmy2-C1R (C1R) is a human lymphoid cell line with low expression of its endogenous class I Ags. These cells and their transfectants expressing HLA-B27 subtypes were cultured in DMEM (Life Technologies, Paisley, U.K.) with 7.5% heat-inactivated FCS. T2 is a TAP-deficient human cell line of lymphoid origin (23). The B*2705-T2 transfectant was a kind gift of Dr. David Yu (University of California, Los Angeles, CA). It was cultured in DMEM supplemented with 5% FCS. RMA-S is a TAP-deficient mutant cell line derived from the murine H-2b lymphoma RBL-5. These cells and their transfectants expressing HLA-B27 subtypes plus human ß2-microglobulin (6, 24, 25, 26) were cultured in RPMI 1640 supplemented with 10% FCS. When cultured at 26°C, T2 and RMA-S cells express class I molecules presumably devoid of peptides. These molecules are unstable at 37°C, but their surface expression at this temperature can be stabilized by exogenous peptide ligands.
Isolation of HLA-B27-bound peptides
About 1–1.5 x 1010 B*2705-C1R cells were lysed at 4°C in 20 mM Tris-HCl buffer, 150 mM NaCl, 1% Nonidet P-40, pH 7.5, containing the following protease inhibitors: 10 µg/ml leupeptin, 2 µg/ml pepstatin, 2.5 µg/ml aprotinin (all from Boehringer Mannheim, Mannheim, Germany), 18.5 µg/ml iodoacetamide, 1 mM EDTA, 2 mM PMSF, 258 µg/ml 1,10 phenantroline (Sigma, St. Louis, MO), and 0.2% sodium azide. Cell lysates were centrifuged, and the supernatant was filtered, precleared through a CNBr-activated Sepharose 4B column (Pharmacia, Uppsala, Sweden), and subjected to affinity chromatography using the W6/32 mAb (IgG2a, specific for a monomorphic HLA-A, -B, and -C determinant). HLA-B27-bound peptides were eluted from the column with 0.1% TFA in water at room temperature, filtered through Centricon 3000 (Amicon, Beverly, MA), and concentrated to 500 µl for fractionation by HPLC. This was conducted in a 2 x 150-mm Deltapack C18 (300Å) column (Waters, Mildford, MA), at a flow rate of 100 µl/min, as follows: isocratic conditions with buffer A (0.1% TFA in water) for 20 min, followed by a linear gradient of 0–44% buffer B (80% acetonitrile, 0.1% TFA in water) for 80 min, and a linear gradient of 44–100% buffer B for another 40 min. Peptide fractionation was simultaneously monitored at 210 and 280 nm, using a Waters 991 photodiode array detector. Fractions of 50 µl were collected and stored at -20°C.
Mass spectrometry analysis and peptide sequencing
The peptide composition of individual HPLC fractions was
determined by matrix-assisted laser desorption/ionization time of
flight (MALDI-TOF) mass spectrometry. A calibrated Reflex instrument
(Brucker-Franzen Analytik, Bremen, Germany) operating in the positive
ion reflectron mode was used. Five-microliter aliquots of a given HPLC
fraction were dried, resuspended in 2 µl of 0.1% TFA in
water:acetonitrile, 2:1, and mixed with 2 µl of saturated
-cyano-4-hydroxycinnamic acid matrix in 0.1% TFA in a
water:acetonitrile ratio of 2:1. One microliter of the mixture was
dried and subjected to analysis.
Peptide sequencing was conducted in a LCQ electrospray/ion trap mass spectrometer (Finnigan MAT, San Jose, CA), equipped with a microspray probe. HPLC fractions were dried down and resuspended in 5 µl methanol/water (1:1) containing 0.1% formic acid. One microliter of this solution was used for analysis. Accurate peptide mass and charge of ionic species were determined by performing "Zoomscan" spectra. This is a high resolution scanning method in which a precursor ion window is selected to incorporate several isotopomers. The charge states of individual product ions were determined at enhanced resolution by scanning across a limited mass/charge (m/z) range. Collision energy and precursor ion resolution were optimized for each individual peptide to obtain the optimum fragmentation spectra. Putative peptide sequences were obtained by database comparison of the fragmentation spectra using the PEPSEARCH program (Bioworks package; Finnigan) in conjunction with the nr-BLAST database from the National Center for Biotechnology Information (National Institutes of Health, Bethesda, MA), followed by manual assignment of expected fragments from the highest-score sequences. Sequence assignments were confirmed by comparing the fragmentation spectra with those of the corresponding synthetic peptides.
In one case, peptide sequencing was conducted by Edman degradation in an ABI 473A automated sequencer (Applied Biosystems, Foster City, CA). A single HPLC fraction of the B*2705-bound peptide pool from 1010 C1R transfectant cells, containing about 10 pmol of the major peptide species, was used.
Peptide synthesis
Peptides were synthesized, using the standard F-moc or the t-BOC chemistries, and purified by HPLC. The correct composition and molecular mass of purified peptides were confirmed by amino acid analysis using a 6300 amino acid analyzer (Beckman, Palo Alto, CA), which also allowed their quantification, and by electrospray/ion trap mass spectrometry, respectively.
Epitope stabilization assay
The quantitative epitope stabilization assay used was previously
described (25). Briefly, RMA-S transfectants were incubated at 26°C
for 24 h. They were then incubated 1 h at 26°C with
10-4 to 10-9 M peptides without FCS,
transferred to 37°C, and collected for flow microcytometry analysis
with the ME1 mAb (IgG1, specific for HLA-B27, B7, B22) after 4 h
for B*2705 and B*2706, or after 2 h for all other HLA-B27
subtypes. Binding was expressed as the C50, which is the
molar concentration of the peptide at 50% of the maximum fluorescence
obtained with that peptide in the concentration range used. Relative
binding of multiple peptides was assessed as follows. First, the
C50 of the reference peptide was calculated. Second, the
concentration of each other peptide required to obtain the fluorescence
value at the C50 of the reference peptide was found by
interpolation. This was designated as EC50, which is equal
to the C50 for the reference peptide. Relative binding was
expressed as the ratio between the EC50 values of the
peptides compared. Peptides with EC50 
5 µM were
considered to bind with high affinity. EC50 values between
5 and 50 µM were considered to reflect intermediate affinity.
EC50 
50 µM indicated low affinity.
Peptide sensitization and other cytotoxicity assays
T2 or RMA-S transfectant cells were incubated for 24 h at 26°C in RPMI 1640 medium supplemented with 10% FCS, in the absence of peptide. Cells were then labeled for 90 min at 37°C with 50 µCi of 51Cr, washed (four times), resuspended in the same medium with 1% FCS, seeded in 96-well plates, and incubated for 30 min at room temperature with synthetic peptides or aliquots of particular HPLC fractions. Effector cells were then incubated with peptide-sensitized targets for 5 h at 37°C at given E:T ratios, and the supernatants were subjected to gamma counting. Percentage of specific 51Cr release was calculated as follows: (experimental lysis - spontaneous lysis)/(maximum release - spontaneous lysis) x 100.
A 4-h 51Cr release assay was used for other target cells, as described above, except that cells were not preincubated with exogenous peptide, unless otherwise specified, or at lower temperature.
Molecular modeling
The RRFFPYYV and RRFFPYYVY peptides in complex with B*2705
were simulated by molecular dynamics, using the AMBER 5.0 package (27)
with the AMBER95 force field (28). Initial coordinates for the Ag
binding domain (residues 1–182) were taken from the crystal structure
of HLA-B*2705 (29) deposited in the Protein Data Bank. Both peptides
were built by assembling two fragments (P1-P3, P4-P
) generated from
a crystal structure database of class I MHC-bound peptides. The P1-P3
part (RRF) was built from the B*2705-bound ARAAAAAAA peptide (29).
Rotameric states for the Arg1 and Phe3 side chains were assigned
according to the structures of RGYVYQGL in complex with
H-2Kb (30), and of LLFGYPVYV in complex with
HLA-A*0201 (31). The C-terminal part (P4-P) has a less-defined
conformation depending on its sequence and length (31). Thus, it was
built from the structures of two class I MHC-bound peptides, an octamer
and a nonamer, with Pro at P5. The FPYYV sequence (P4-P8) of the
octamer was constructed from VPLRPMTY in complex with
HLA-B*3501 (32). For the nonamer, LLFGYPVYV in complex with
HLA-A*0201 (31) was used as a template to build the C-terminal FPYYVY
sequence (P4-P9). Residue changes in P4 to P-1 were introduced using
the SYBYL modeling package (Tripos Associates, St. Louis, MO) without
altering the dihedral angle of the side chains. The C-terminal side
chain (Val8 or Tyr9) was orientated according to the two MHC-bound
peptides described above (LLFGYPVYV, VPLRPMTY).
The N- and C-terminal fragments were connected to define a
trans peptide bond between P3 and P4. After adding all
hydrogen atoms, both B*2705-peptide complexes were energy minimized in
vacuum, as previously described (33, 34). To sample the broadest
conformation space in the bound state, the peptide was then annealed to
1000 K while maintaining the protein fixed, according to a described
procedure (35). The MHC-peptide complex was then solvated in a shell of
1350 water molecules, minimized, and subjected to a 200-ps
restrained molecular dynamics simulation, as previously reported (35).
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Results |
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The peptide dependency of CTL 27S69 was suggested by failure to
lyse B*2705-T2 cells. In a first attempt to determine the epitope
recognized by this CTL, the B*2705-bound peptide pool from about
1010 C1R transfectant cells was fractionated by HPLC (Fig. 1
A). Aliquots of individual
fractions were incubated with B*2705-T2 transfectants, and tested for
lysis by the CTL clone. A peak of sensitizing activity spanning HPLC
fractions 205–208 (Fig. 1B) suggested that CTL 27S69
recognized a peptide eluting in these fractions. Thus, their
composition was determined by MALDI-TOF mass spectrometry. Fractions
205 and 206, which showed the highest sensitizing activity, contained
at least 15 and 7 molecular species, respectively. The amino acid
sequences of two of the four peptides that eluted in both of these
fractions were determined by electrospray/ion trap mass spectrometry.
The sequence of the RRFFPYYV octamer (molecular mass: 1149 Da), a
proteasome C5 subunit-derived peptide, was determined from its MS/MS
spectrum (Fig. 2), as follows: matching
of the observed pattern of product ions using the PEPSEARCH program in
conjunction with the nr-BLAST database suggested the sequence mentioned
above. Visual inspection confirmed that the data were consistent with
this assignment (Fig. 2), and comparison with the product ion
spectrum of the corresponding synthetic peptide provided confirmation
of the structure. A minor signal corresponding to the same peptide was
detected in fraction 207. The second sequence (molecular mass: 1159 Da)
was RRLPIFSRL, a peptide previously identified as a natural ligand of
B*2705 (36) and B*2709 (37).
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, and was previously identified as
a natural ligand of B*2701 (26), B*2703, and B*2705 (38). The two peptides sequenced from fractions 205 and 206 were synthesized and tested for the capacity to sensitize B*2705-T2 cells for lysis by CTL 27S69. In an initial experiment, the RRFFPYYV octamer induced significant lysis even at 20 nM concentration, whereas RRLPIFSRL did not induce lysis at 200 µM. This result indicates that the peptide epitope recognized by CTL 27S69 in the context of B*2705 is the RRFFPYYV octamer.
CTL 27S69 distinguishes between closely related natural ligands
That HPLC fraction 208 sensitized poorly B*2705-T2 transfectants
for lysis (Fig. 1B) suggested that its predominant peptide
RRFFPYYVY was inefficiently recognized by CTL 27S69. Thus, the capacity
of this CTL clone to distinguish between this peptide and the RRFFPYYV
octamer was quantitatively analyzed. The octamer efficiently sensitized
B*2705-T2 (Fig. 3A) and
B*2705-RMA-S (Fig. 3B) cells for lysis: half-maximal lysis
was obtained at 1.8 x 10-8 M and 1.4 x
10-7 M, respectively. This difference is consistent with
the lower avidity of human CTL for murine, relative to human target
cells. The nonamer was recognized about 100- or 1000-fold less
efficiently on B*2705-T2 and B*2705-RMA-S targets, respectively. This
was not due to lower binding of the nonamer: actually, this peptide
bound better than the octamer to B*2705-RMA-S cells (Fig. 3C). The nonamer was about fourfold more abundant than the
octamer in the B*2705-bound peptide pool, as estimated from the
respective intensity peaks in the MALDI-TOF mass spectra of all of the
HPLC fractions containing either peptide. This correlates with their
relative binding to B*2705 in vitro.
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CTL 27S69 cross-reacts with B*2702 and B*2703 through recognition of the same peptide as in B*2705
CTL 27S69 cross-reacts with B*2702 and B*2703 on LCL (20). Thus,
binding of the RRFFPYYV octamer and the corresponding nonamer, and
their recognition in the context of these two subtypes were tested.
Both peptides bound to B*2702 (Fig. 4A) and B*2703 (Fig. 4B), with EC50 values in the range commonly
found among natural ligands. Indeed, the nonamer was sequenced from the
B*2703-bound peptide pool (38). In addition, CTL 27S69 efficiently
killed B*2702-RMA-S targets sensitized with the octamer (half-maximal
lysis at 0.8 x 10-7 M) and, about 70-fold molar less
efficiently, with the nonamer (Fig. 4C). The octamer, but
not the nonamer, was also recognized in the context of B*2703
(half-maximal lysis at 4.6 x 10-7 M) (Fig. 4D).
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B*2704, B*2706, and B*2710 bind, but do not present, the peptide epitope to CTL 27S69
This CTL clone failed to lyse LCL expressing B*2704 or B*2706
(20), as well as B*2710-C1R cells (data not shown). All three subtypes
have Glu152, instead of Val152, which is present in all other HLA-B27
subtypes. This is the only change between B*2710 and B*2705. Binding to
B*2704-, B*2706-, or B*2710-RMA-S cells, and lysis of these
transfectants in the presence of the peptide epitope were analyzed
(Fig. 5). Both the RRFFPYYV octamer
and the corresponding nonamer bound in vitro to B*2704,
B*2706, and B*2710 with EC50 values in the range of natural
ligands (Fig. 5A–C). In addition, the RRFFPYYV
octamer was identified and sequenced from a HPLC fraction of
B*2704-bound peptides, by electrospray/ion trap mass spectrometry (data
not shown). In spite of the binding observed, CTL 27S69
failed to lyse B*2704-, B*2706-, or B*2710-RMA-S cells even
at the highest peptide concentration used (Fig. 5D). The
octamer also failed to sensitize C1R transfectants expressing these
subtypes (data not shown).
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B*2701 presents exogenous, but not the endogenous peptide epitope to CTL 27S69
CTL 27S69 did not lyse B*2701-positive LCL (20), or B*2701-C1R
transfectants. As in B*2705, the nonamer, which is a natural B*2701
ligand (26), bound better than the octamer to B*2701-RMA-S cells (Fig. 6A). Although the
EC50 of the octamer was also in the range of natural
ligands, it was not found in the corresponding HPLC fractions from the
B*2701-bound peptide pool, upon analysis by electrospray/ion trap mass
spectrometry. Other peptides coeluting with the octamer in the
B*2705-bound peptide pool were also in the fractions analyzed from
B*2701 (data not shown). This suggests that the octamer is not bound in
vivo to B*2701. Thus, failure of CTL 27S69 to cross-react with B*2701
LCL is probably due to absence of the peptide epitope on this subtype.
In peptide sensitization assays, significant lysis of B*2701-RMA-S
cells was obtained with the octamer (half-maximal lysis at
10-6 M) and, about 100-fold lower, with the nonamer (Fig. 6B). In addition, B*2701-C1R cells were highly sensitized
with the octamer (half-maximal lysis at 1.7 x 10-8
M) (Fig. 6C). These results indicate that RRFFPYYV is
efficiently recognized by CTL 27S69 in the context of B*2701, further
supporting that lack of cross-reaction with this subtype is due to lack
of constitutive binding of the peptide to B*2701 in vivo.
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The molecular basis for the capacity of CTL 27S69 to distinguish
between the RRFFPYYV and RRFFPYYVY ligands was addressed by molecular
modeling of both peptides in complex with B*2705. They were stabilized
in the peptide-binding groove by numerous hydrogen bonds and nonpolar
interactions. However, whereas hydrogen bonding was quantitatively
similar in both complexes, the nonamer established significantly more
nonpolar contacts (total of 72) than the octamer (total of 53), mainly
because of additional interactions of Tyr9 in the F pocket. The total
buried surface areas for the octamer and the nonamer bound to B*2705
were 732 and 812 Å2, respectively. These results are
consistent with the better binding of the nonamer to B*2705 (Fig. 3).
The differential recognition of the octamer and nonamer by CTL 27S69
can be explained by the different conformations of both peptides in
complex with B*2705 (Fig. 7). Although
the accessible surface area was only slightly higher for the octamer
(539 Å2) than for the nonamer (506 Å2), the
conformation of the main chain was different, especially in the central
part of the sequence (P4-P5), where it bulged out more prominently in
the nonamer. Besides Arg1, which was partially accessible and similarly
oriented in both peptides, three other side chains (Phe4, Pro5, and
Tyr7) were accessible for the octamer, and four (Phe4, Pro5, Tyr7, and
Val8) for the nonamer. Phe4 was rather similarly oriented in both
cases. In contrast, Pro5 bulged out much more for the nonamer. In
addition, a major qualitative difference occurred for Tyr7. In the
nonamer, its side chain was located in a three-dimensional space not
occupied by any atom of the bound octamer. Val8 was exposed for the
nonamer. In the octamer, this residue is directed inward into the F
pocket in the same way as Tyr9 in the nonamer, although the larger Tyr9
side chain gets deeper into this pocket.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The RRFFPYYV octamer derives from the C5 subunit of the proteasome,
which is abundant in the cell (44), and was recovered from the
B*2705-bound peptide pool in relatively substantial amounts. However,
both abundant (9) and nonabundant (8, 11) peptides can stimulate
alloreactive CTL. This epitope probably has high affinity for the TCR
of CTL 27S69, since this clone can efficiently lyse murine B*2705-P815
(our unpublished results) and, in the presence of added peptide, RMA-S
transfectant cells. Both TCR affinity and expression level of this
peptide may contribute to its alloimmunogenicity. A minimum threshold
of avidity is probably required to stimulate a given allo-CTL
precursor. This threshold could be reached at low determinant density
through high TCR affinity, or with high epitope expression in cases of
lower affinity. Since serial TCR engagement by relatively few
MHC-peptide complexes may be a mechanism of T cell triggering (45),
nonabundant peptides may be allostimulatory. The requirements for
alloimmunogenicity are important because they dictate the diversity of
peptide epitopes in an allospecific T cell response. For instance,
among the many class I-bound peptides involved in alloreactivity (1, 8), some can be immunodominant (13). The RRFFPYYV peptide was not
recognized by three other anti-B*2705 clones tested,
including one from the same donor (data not shown), but we cannot rule
out that it might be one of a relatively limited set of peptides
driving the anti-B*2705 response. This was suggested by
restrictions in N+Dß and J usage among B27-allospecific TCR (46).
Two aspects of this study provide novel insights into the molecular basis of T cell allorecognition. One is that another natural ligand closely related to the octameric epitope allowed us to assess the capacity of a CTL clone to distinguish between similar peptides in an allo-MHC context. The second aspect is the knowledge of the fine specificity of the CTL clone with HLA-B27 subtypes (20). This allowed us to analyze the relationship between expression and recognition of the peptide epitope in the context of different allo-MHC molecules, and therefore to assess the role of HLA polymorphism in allorecognition, aside from its effects on peptide binding.
Although self-restricted CTL are sensitive to subtle peptide changes, the capacity of alloreactive CTL to distinguish between related ligands could be limited by a more prominent contribution of MHC residues to the allospecific determinant. Cross-reaction of CTL 27S69 with the RRFFPYYVY nonamer might be due, on the basis of modeling, to conservation of a portion of the epitope involving the MHC molecule and the peptidic Arg1 and Phe4. However, conformational and accessibility differences in the P5-P8 region explain the better recognition of the octamer. Thus, the different alloantigenicity of these highly similar ligands illustrates the critical role of peptide conformation in alloreactivity.
CTL cross-reacting with other MHC Ags do not necessarily recognize the
same peptide in the various contexts. For example, the murine
alloreactive CTL clone 2C recognized three unrelated peptides in the
context of Ld, Kbm3, and, in self-restricted
fashion, Kb, respectively (9, 17, 18). Although CTL
cross-reactions with HLA-B27 subtypes were assumed to reflect
similarities among subtype-bound peptide repertoires (20, 47),
recognition of the same peptide by CTL 27S69 on B*2705, B*2702, and
B*2703 provides the first formal demonstration of this assumption. It
also indicates that the changes between B*2702 or B*2703 with B*2705
(Y
H59 in B*2703; DN77, TI80, and LA81 in B*2702) are
not critical for this epitope. Presentation of the same viral peptide
to B*2705-restricted CTL by B*2702 has been shown for EBV-specific T
cells (48, 49). However, 7 of 10 B*2705-restricted CTL clones failed to
recognize the viral epitope in the context of B*2702 (49). Thus,
although not for CTL 27S69, polymorphic B*2702 residues can alter some
T cell epitopes without impairing binding of the corresponding peptide.
From the results with B*2702 and B*2703, it should not be concluded
that, upon expression of the relevant peptide, the structure of the
alloantigen is not critical for allorecognition. Indeed, in spite of
good binding, the RRFFPYYV octamer was not recognized in the B*2704,
B*2706, or B*2710 context. Direct impairment of TCR binding by the E152
residue in these three subtypes is strongly supported by the fact that
this is the only change between B*2710 and B*2705. We have reported
recently that, in spite of little cross-reaction of anti-B*2705 CTL
with B*2710, both subtypes bind in vivo similar peptide repertoires.
Molecular modeling further suggested that the E152 change in B*2710 did
not alter the conformation of bound peptides, but directly impaired TCR
interaction (35). CTL 27S69 now provides the first example of an
anti-B27 alloreactive CTL clone that fails to recognize its peptide
epitope across the VE152 change. Therefore, besides the peptide, the
structure of HLA-B27 directly and critically contributes to the
allospecific epitope. The contribution of the MHC molecule to
allorecognition is likely to severely limit cross-reactivity between
class I alloantigens that bind common peptides (50).
Cross-reaction of CTL 27S69 with B*2701 only in the presence of exogenous peptide strongly suggests that the octamer epitope is not endogenously presented by this subtype, in spite of significant binding in vitro. This was supported by failure to detect the octamer in the B*2701-bound peptide pool by mass spectrometry. Since B27-bound peptides were isolated from C1R transfectants both for B*2701 and other subtypes, it is extremely unlikely that the octamer is not generated in B*2701-C1R transfectants due to a defect of these cells, independently of B*2701. Since the octamer bound similarly in vitro to B*2701 and B*2705 (EC50 7 and 8 µM, respectively), it also seems unlikely that failure to bind in vivo could be due to disadvantageous competition with other ligands for binding to B*2701, relative to B*2705. A possibility that we favor is that the octamer might be generated by trimming of the HLA-B27-bound nonamer, rather than by proteasome-mediated cleavage, and that in B*2701 such trimming is impaired. This is suggested by a report that MHC class I molecules influence the precise structure of endogenous ligands (51). Since the RRFFPYYVY nonamer is naturally presented by B*2701 (26), lack of cross-reaction with B*2701 indicates that the avidity of CTL 27S69 for the constitutive B*2701 + nonamer complex is insufficient for lysis.
In conclusion, identification of a peptide epitope involved in HLA-B27 alloreactivity allowed us to establish that T cell allorecognition is very dependent both on the precise structure of the peptide, as to discriminate between closely related natural ligands, and on the structure of the MHC molecule. This may either influence expression of the peptide at the cell surface, or directly impair TCR binding.
The findings reported in this work have implications for the pathogenetic role of HLA-B27 in ankylosing spondylitis and other spondyloarthropathies. Among other possible mechanisms, the arthritogenic peptide hypothesis proposes that peptide(s) presented by HLA-B27 would be recognized by autoreactive CTL activated upon external challenge, such as a bacterial infection (52). This hypothesis must explain that multiple subtypes, such as B*2705, B*2702, and B*2704, are associated with ankylosing spondylitis (53), whereas B*2706 and B*2709 are less or not associated with this disease (54, 55). Presumably, disease-associated subtypes should present some common peptides to CTL. Our results demonstrate that B*2705 and B*2702 present the same peptide to an alloreactive CTL, as they do to some EBV-restricted CTL clones (49). A potential problem arises from the fact that B*2704, although it binds some of the same ligands as B*2705 and B*2702 (25, 56), cannot present those that have been tested to the same CTL, as shown in this work and for EBV epitopes (48, 49). Cross-reaction of some anti-B*2705 alloreactive CTL clones with B*2704 or with B*2704 plus B*2702, respectively (20), suggests that, in spite of residue 152, some peptides might be presented by all three B*2705, B*2702, and B*2704 subtypes to the same CTL. However, this awaits molecular identification of the corresponding peptide epitopes. There are at least two possibilities that make compatible the peptide-presenting properties of these three subtypes with their association to spondyloarthropathy. One is that putative arthritogenic peptides may be different for different subtypes. A second possibility, that might seem more likely, is that the same peptide is recognized in the context of different subtypes by arthritogenic CTL.
Although, obviously, alloreactive CTL are not related to B27-mediated spondyloarthropathy, their recognition of a same peptide in the context of different subtypes illustrates the extent to which HLA-B27 subtypes may interchangeably act as restriction elements for given peptides. This feature would be critical for a putative pathogenetic role of HLA-B27 as a peptide-presenting molecule.
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2 The contribution of A.P. and M.G.-P. to this work is equal.
3 Address correspondence and reprint requests to Dr. José A. López de Castro, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Facultad de Ciencias, Cantoblanco, 28049 Madrid, Spain. E-mail address:
4 Abbreviations used in this paper: LCL, lymphoid cell line; TFA, trifluoroacetic acid; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight.
Received for publication April 30, 1998. Accepted for publication July 14, 1998.
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ß T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex. Science 274:209.2 helix of MHC class I is revealed by the crystal structure of HLA-B*3501. Immunity 4:203.[Medline]
and ß chains. Eur. J. Immunol. 25:2479.[Medline]
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) peptides by CTL
27S69. The B*2705 natural ligand RRYQKSTEL (
) was used as negative
control. Half-maximal lysis was obtained at 1.8 x
10-8 M of the octamer. The same lysis required 2.5 x
10-6 M of the nonamer. Specific lysis of B*2705-C1R cells
at the E:T ratio used (0.5:1) was 49%. B, Lysis of
B*2705-RMA-S target cells sensitized with RRFFPYYV, RRFFPYYVY, or the
negative control RRYQKSTEL by CTL 27S69. Half-maximal lysis was
obtained at 1.4 x 10-7 M of the octamer. The same
lysis required more than 10-4 M of the nonamer. Specific
lysis of B*2705-C1R cells at the E:T ratio used (0.75:1) was 45%.
C, Binding of RRFFPYYV, RRFFPYYVY, and KTGGPIYKR as
negative control to B*2705 on RMA-S transfectant cells.
EC50 was 2 and 8 µM for the nonamer and the octamer,
respectively. Data are means of two experiments. For experimental
details, see Materials and Methods.
), B*2704 (•), B*2706
(
), and B*2710 (
) sensitized with the synthetic RRFFPYYV
octamer, by CTL 27S69. E:T ratio was 0.75:1, 1:1, 1.25:1, and 1:1,
respectively. Specific lysis of the control B*2705-C1R cells in these
experiments was 45, 71, 71, and 58%, respectively. Data are means of
two experiments. For experimental details, see Materials and
Methods.
