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Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel
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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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A cascade of events can influence which peptides are found on the cell surface, including 1) the presence of endogenous and exogenous proteins; 2) the appropriate degradation of these proteins in intracellular compartments; 3) the ability of the degraded peptides to bind in the groove of the particular HLA molecules; and 4) the successful transport of these molecules to the cell surface. The absence or dysfunction of any of these processes can lead to defective peptide presentation on the tumor cell surface.
The interaction of the three-dimensional structure of the peptide-binding groove of the class I (and II molecules) with the conformation of the associated peptide determines which peptides can bind to particular HLA molecules as well as the affinity of this interaction. Specific sites in the groove of HLA molecules (called anchor sites) play a major role in determining the binding of the peptide to the MHC molecule. Other amino acid residues are more involved in interacting with the TCR, leading to recognition of this peptide and the MHC complex (1, 2, 3, 4, 5, 6). The analysis of known peptides recognized by T cells as well as the analysis of sequences of pooled peptides eluted from MHC molecules have helped in characterizing peptides associated with particular MHC molecules (5, 6). The relatively uniform length of these peptides (e.g., 8–10 aa for most class I molecules and slightly longer peptides for class II molecules) has enabled defining of these allele-specific motifs for most of the common class I (and II) molecules (1, 2, 3, 4, 5, 6). From these and other studies, it was determined that position 2 was an anchor residue favoring a leucine or methionine, and position 9 was a second anchor residue favoring the binding of a valine or leucine to the HLA-A*0201 molecule. Other residues with a lesser degree of preferred binding to HLA-A2 were also found. Examples of predominantly anchor-binding residues for other HLA alleles were also identified (3, 5). Knowledge of these allele-specific motifs has facilitated the identification of the immunogenic peptides of tumor Ags (7, 8). Most tumor Ags that have been characterized were derived from melanomas (9, 10, 11). These studies revealed that in contrast to most immunogenic viral peptides that have high binding affinity for their corresponding MHC molecules, most of the immunogenic melanoma and melanocyte differentiation Ag peptides have a moderate or relatively low affinity for the HLA-A*0201 molecule (12, 13, 14, 15). For example, of the four immunodominant HLA-A*0201-restricted peptides derived from the MART and gp100 Ags, three of the four peptides have a suboptimal amino acid residue at anchor position two and the other peptide has a suboptimal residue at anchor position nine (15). It is likely that T cell precursors capable of binding peptides with high affinity for HLA-A*0201 are deleted during lymphocyte maturation in the thymus and that only those T cells with relatively weak recognition of peptides survive negative selection (16, 17). Thus, although a correlation has been demonstrated between immunogenicity and peptide-binding affinity to class I MHC molecules for peptides derived from viral Ags, this appears not to be the case for the immunogenicity of peptides derived from nonmutated self determinants (12, 13, 14, 15). The relatively low binding affinity of the immunodominant self peptides suggested that amino acid substitutions at anchor residue positions could increase the binding affinity without interfering with peptide recognition, and thus increase the immunogenicity of the peptide (18, 19, 20, 21). For example, Parkhurst et al. (15) studied numerous synthetic peptides with one or two amino acid substitutions of gp100-derived peptides designed to increase the binding affinity to the HLA-A*0201 molecule (15). In the gp100:209–217 peptide, the relative binding affinity of the leucine modification instead of threonine at the second position resulted in a peptide with a 52-fold higher binding affinity than the native peptide and yet was recognized as well as the native peptide by a specific tumor-infiltrating lymphocyte (TIL). CTLs induced by in vitro sensitization using the modified peptide exhibited superior recognition of the native peptide compared with CTLs raised with the native peptide. Similarly, a peptide with a modification of methionine at the second position was more effective than the native peptide in inducing CTLs reactive with the native peptide in vitro.
Based on these in vitro studies, a pilot clinical protocol was performed in which HLA-A*0201 patients with metastatic melanoma were immunized with the gp100:209–217 (G9-209 M)-modified peptide (M substituted for T at the second anchor position, aa 210) (22, 23). Using a stringent in vitro assay comparing pre- and postimmunization samples after a single in vitro exposure to peptide, only two of eight patients immunized with the native peptide were successfully immunized compared with 10 of 11 patients immunized with the modified peptide (23).
Interestingly, recent studies demonstrated changes in the fine specificity of gp100-derived 209-reactive T cells in some patients, following their vaccination with the 209 M-modified peptide (24). Moreover, it was shown that some PBMC-derived cloids isolated from these patients recognize the G9-209 M-modified peptide, but not the native one (24). These results suggested that a modification at an MHC anchor position may influence the overall conformation of the MHC-peptide complex groove and that T cells may sometimes recognize these differences.
Recently, we were able to isolate a new class of human recombinant Abs that recognize tumor-associated MHC-peptide complexes with the same specificity as the TCR. Interestingly, they recognized the MHC-peptide complex only when the specific peptide was present in the complex, yet unlike TCRs, they did so with an affinity in the nanomolar range (25, 26). In fact, we were able to isolate a surprisingly large panel of these human recombinant Fab Abs that exhibit a characteristic TCR-like binding specificity to each of three gp100-derived epitopes. We isolated these from a large nonimmune repertoire of phage Fab Abs selected on recombinant HLA-A2 complexed with three common antigenic T cell HLA-A2-restricted epitopes derived from the melanoma differentiation Ag gp100. These TCR-like Abs recognize the native MHC-peptide complex expressed on the surface of APCs. Moreover, they can detect the specific MHC-peptide complexes on the surface of melanoma tumor cells (26). The gp100-derived 209-modified epitope, 209 M, was one of the peptides used to select such Abs. In this study, we describe the binding characteristics of these TCR-like Abs and show that a TCR-like Ab selected toward the 209 M-modified peptide was able to bind only to HLA-A2 complexes displaying the modified, but not the native peptide. In this way, we demonstrate that these Abs can be used as an excellent molecular tool to study the structural and functional consequences of modifying MHC anchor residue positions. Importantly, we show in this study using these TCR-like Abs as conformation-sensitive sensors that a modification in anchor position can dramatically influence the conformation of the HLA-A2-peptide complex molecule.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2
microglobulin (2m)-HLA/A2 (scMHC)/peptide complexes
scMHC/peptide complexes were produced by in vitro refolding of
inclusion bodies produced in Escherichia coli as described
(27). Briefly, a scMHC construct, in which the
2m and HLA-A2 genes are connected to each
other by a flexible peptide linker, was designed to contain the BirA
recognition sequence for site-specific biotinylation at the C terminus
(scMHC-BirA). This construct is expressed in E. coli,
and upon induction with isopropyl
-D-thiogalactoside, intracellular inclusion
bodies that contain large amounts of the recombinant protein
accumulate. Inclusion bodies are purified, reduced, and subsequently
refolded in a redox-shuffling buffer system (0.1 M Tris, 0.5 M
arginine, 0.09 mM oxidized glutathione, pH 8.0) in the presence of a
5–10 molar excess of the antigenic peptides. Correctly folded
MHC/peptide complexes were isolated and purified by anion exchange
Q-Sepharose chromatography (Amersham Pharmacia Biotech,
Piscataway, NJ). Filtration using Centricon-30 units (Amicon, Bedford,
MA) was used to exchange the elusion buffer with Tris-HCl (10
mM, pH 8.0) and concentrate the scMHC-peptide complex to 1 mg/ml for
specific biotinylation using the BirA enzyme (Avidity, Denver, CO) as
previously described (28, 29). Excess biotin was removed
from biotinylated complexes using a G-25 desalting column. The
homogeneity and purity of the scMHC-peptide complexes was analyzed by
various biochemical means including SDS-PAGE, size exclusion
chromatography, and ELISA as described previously (30).
The biological function of the scMHC-peptide complexes was determined
by the ability of tetramers to stain CTL lines and clones in a
peptide-specific manner. The generation of the scMHC-peptide tetramers
and CTL staining procedures have been previously described in detail
(28).
Isolation of TCR-like Abs
A large human Fab library containing 3.7 x 1010 different Fab clones was used for the selection (30). Phages (1013) were first preincubated for 1 h at room temperature (RT) in PBS containing 2% nonfat dry milk with streptavidin-coated paramagnetic beads (200 µl; Dynal Biotech, Oslo, Norway) to deplete streptavidin binders. Streptavidin-coated paramagnetic beads (200 ml; Dynal Biotech) were also incubated in PBS + 2% milk for 1 h at RT. The remaining phages were subsequently incubated for 1 h with decreasing amounts of biotinylated scMHC-peptide complexes (500 nM for the first round and 100 nM for the following rounds). Streptavidin magnetic beads were added, and the mixture was incubated for 15 min with continuous rotation. A magnetic force was applied to pull down phages bound to biotinylated complexes. After 10 washes of the streptavidin-bound complexes with PBS/0.1% Tween 20 and two washes with PBS, the bound phages were eluted by incubation for 5 min with 1 ml of triethylamine (100 mM). The elusion mixture was neutralized by the addition of 100 µl of Tris-HCl (1 M, pH 7.4) and used to infect E. coli TG1 cells (OD600 = 0.5) for 30 min at 37°C. Bacteria were grown overnight at 30°C on 2YT plates containing 100 µg/ml ampicillin (2YT/A/G) and 2% glucose.
Colonies were collected from the plates in 2YT/A/G and diluted 1/100 in 50 ml of medium. Cells were grown to OD600 = 0.5, and M13KO7 helper phage (5 x 1011 CFU) was added to 5 ml of the culture. After incubation at 37°C for 30 min, the cells were centrifuged, resuspended in 25 ml of 2YT, ampicillin (100 µg/ml), kanamycin (50 µg/ml), and grown overnight at 30°C. Phages were collected from culture supernatants and purified for the next round of panning by polyethylene glycol precipitation. The diversity of the selected Abs was determined by DNA fingerprinting. The Fab DNA of different clones was PCR amplified using the primers pUC-reverse (5'-AGCGGATAACAATTTCACACAGG-3') and fd-tet-seq24 (5'-TTTGTCGTCTTTCCAGACGTTAGT-3'). The resulting PCR fragments were digested with BstNI (NEB, Beverly, MA) (2 h, 37°C) and analyzed by agarose gel electrophoresis.
Expression and purification of soluble recombinant Fab Abs
Soluble Fabs were purified from the periplasmic fraction of BL21
cells using the hexahistidine tag fused to the CH1 domain of the Fabs.
We produced and analyzed two to four Fab clones for each complex, which
were selected according to their specificity pattern as assayed by
ELISA with phage and soluble Fab. An overnight starter culture of Fab
specific clones was grown at 30°C. Cells were diluted 1/100 into 500
ml of 2YT/A/G, grown to OD600 = 0.8–1.0, and induced
to express the recombinant Fab Ab by the addition of 1 mM isopropyl
-D-thiogalactoside for 4 h at 30°C. The cells
were centrifuged and the pellet was resuspended in 5 ml of a B-PER
solution (Pierce, Rockford, IL) to release periplasmatic content. After
30 min of rotated incubation at RT, the solution was centrifuged
(15,000 rpm, 15 min) and the supernatant was incubated with 0.5 ml of
prewashed TALON beads suspension (Clontech Laboratories, Palo Alto, CA)
for 45 min at RT. The solution was applied onto a Bio-Rad disposable
column (Bio-Rad, Hercules, CA), and after sedimentation, the
beads were washed three times with 10 ml of PBS, 0.1% Tween 20 (pH
8.0). The bound Fabs were eluted using 0.5 ml of 100 mM imidazole in
PBS. The eluted Fabs were dialyzed twice against PBS (overnight, 4°C)
to remove residual imidazole. The homogeneity and purity of the
purified Fabs were determined by analysis on nonreduced and reduced
SDS-PAGE.
ELISA with purified Fab Abs
The binding specificity of individual phage clones and soluble
Fab was determined by ELISA using biotinylated scMHC-peptide complexes.
ELISA plates (Falcon; BD Biosciences, Franklin Lakes, NJ) were
coated overnight with BSA-biotin (1 µg/well). After having been
washed, the plates were incubated (1 h, RT) with streptavidin (1
µg/well), washed extensively, and further incubated (1 h, RT) with
0.5 µg of MHC/peptide complexes. Plates were blocked for 30 min at RT
with PBS/2% skim milk, and subsequently were incubated for 1 h at
RT with phage clones (
109 phages/well) or
various concentrations of soluble purified Fab, and after washing, with
1/1000 HRP-conjugated/anti-myc Ab. Detection was
performed using tetramethylbenzidine reagent (Sigma-Aldrich, St.
Louis, MO). The HLA-A2-restricted peptides used for specificity studies
of the Fab phage clones or purified Fab Abs are as follows: gp100
(154): KTWGQYWQV; gp100 (209): IMDQVPFSV; gp100 (280): YLEPGPVTV;
MUC1: LLLTVLTVL; HTLV-1 (TAX): LLFGYPVYV; MART1 (27): AAGIGILTV; hTERT
(540): ILAKFLHWL; hTERT (865): RLVDDFLLV; p53 (149): STPPPGTRV.
Flow cytometry
The B cell line RMAS-HHD, which is transfected with a sc
2M-HLA-A2 gene (31),
EBV-transformed B-lymphoblast JY cells, or tumor cells as indicated
were used to determine the reactivity of the recombinant Fab Abs with
cell surface-expressed HLA-A2/peptide complexes. About
106 RMAS-HHD cells were washed twice with
serum-free RPMI and incubated overnight at 26°C in medium containing
100 µM concentration of the peptide. JY cells were loaded with
peptide (100 µM) at 37°C. The APCs were subsequently incubated at
37°C for 2–3 h to stabilize cell surface expression of MHC-peptide
complexes. The cells were incubated for 60–90 min at 4°C with
recombinant Fab Abs (10–100 µg/ml) in 100 µl. After three washes,
the cells were incubated with FITC-labeled anti-human Fab (The
Jackson Laboratory, Bar Harbor, ME). After a final wash, the cells were
resuspended in ice-cold PBS.
Adherent tumor cells were harvested by trypsinization and resuspended in cold RPMI. All subsequent washes and incubations were performed in ice-cold PBS as described previously for RMAS-HHD peptide-loaded cells. Analysis of the cells was performed by a FACStar flow cytometer (BD Biosciences, Mountain View, CA), and the results were analyzed with the WinMDI program (J. Trotter, http://www.facs.scripps.edu/).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, we isolated a large panel of human recombinant Fab Abs
that exhibit a characteristic MHC-restricted, peptide-dependent,
TCR-like binding specificity to each of three major gp100-derived T
cell epitopes (9, 10). These Abs were isolated from a very
large naive repertoire (3.7 x 1010
independent clones) of human Fab Abs displayed on phage, selected on
scHLA-A2 complexes containing gp100 peptides (26, 27, 28, 30). gp100 is recognized by many HLA-A2-restricted
melanoma-reactive TILs that have been isolated from melanoma patients
(9, 10, 15). Five T cell epitopes have been identified in
gp100; three of them are common immunogenic epitopes recognized by CTLs
derived from different patients (9, 10): G9154
(KTWGQYWQV), G9209 (ITDQVPFSV), and G9280 (YLEPGPVTA). It has been
demonstrated previously that peptides G9-209 and G9-280 can be modified
at the MHC anchor positions 2 (in G9-209M) and 9 (in G9-280V) to
improve the binding affinity to HLA-A2 (15). When
screening the large Fab phage display library, we isolated Fabs 1A9 and
2F1, which recognize the HLA-A2/G9-209M and HLA-A2/G9-280V MHC-peptide
complexes, respectively, in a TCR-like manner. Thus, as shown in Fig. 1
, A and B, these
recombinant Fab Abs bound to the MHC-peptide complex only when the
specific peptide was present in the complex; yet unlike TCRs, they did
so with an affinity in the nanomolar range (26). As shown
in Fig. 1, C and D, these TCR-like Abs recognize
the native MHC-peptide complex expressed on the surface of
peptide-pulsed APCs. We used two APC systems. The first consists of
murine TAP2-deficient RMA-S cells transfected with the human HLA-A2
gene in an sc format (HLA-A2.1/Db-2m sc)
(RMA-S-HHD cells). gp100-derived or control peptides were loaded on the
RMA-S-HHD cells, and the ability of the selected Fab Abs to bind to
peptide-loaded cells was monitored by FACS. The HLA-A2/G9-209M-specific
Fab Ab 1A9 recognized RMA-S-HHD cells loaded with G9-209M peptide, but
not at all cells loaded with the other two gp100 HLA-A2-restricted
epitopes G9-154 or G9-280V (Fig. 1C), or the other 10
control HLA-A2-resticted peptides (data not shwon). Similar results
were observed in the FACS analysis of the G9-280V-specific Fab Ab 2F1
(Fig. 1D). The Fab Abs were analyzed on RMAS-HHD cells
loaded with 10 different control HLA-A2-restricted peptides including
cross-reaction studies among the gp100-derived peptides, and similar
specificity results were observed. Moreover, RMAS-HHD cells loaded with
the G9-209M epitope reacted only with Fab 1A9 directed toward the
G9-209M-containing complex, but not with Fabs 2F1 or G2D12, which
recognized HLA-A2 in complex with the G9-280V or G9-154 gp100-epitopes,
respectively (Fig. 1E). Similar specificity results were
observed with a second type of APCs tested, namely EBV-transformed B
lymphoblast JY cells, which are TAP+, and
consequently, displaying the exogenous peptide is facilitated by
peptide exchange (data not shown).
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The Fab Abs 1A9 and 2F1 were isolated by selection on recombinant
HLA-A2 complexes displaying the modified peptides G9-209M and G9-280V,
respectively. When we tested the binding of these Abs by ELISA to
HLA-A2 displaying the modified or the wild-type peptide, we observed an
interesting phenomena, namely that Fab 1A9 binds only to the HLA-A2
complex displaying the modified G9-209M, but not the wild-type peptide
G9-209 (Fig. 2A). The binding
specificity of 1A9 is maintained since it did not bind to the control
gp100-derived G9-280 and its modified form G9-280V (Fig. 2A). However, Fab 2F1, selected on HLA-A2 complexes
displaying the modified G9-280V peptide, binds the modified as well as
the native peptide (Fig. 2B), but not to complexes
displaying the G9-209 or G9-209M epitopes. The differential binding of
Fab 1A9 is not due to differences in the stability of the
HLA-A2/peptide complex during the assay, because as shown (Fig. 2, C and D), the conformation-specific mAb w6/32,
which binds HLA molecules only when folded correctly and when it
contains peptide, bound to all four complexes with a similar intensity
(Fig. 2C). Titration binding of mAb w6/32 to HLA-A2/peptide
complexes generated with wild-type 209 and modified 209-2M peptide did
not reveal any differences in complex stability as well (Fig. 2D).
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To demonstrate that the differential recognition of modified vs
wild-type peptide by 1A9 can be observed also at the level of the
native complexes, as displayed on the surface of cells, we tested the
binding of 1A9 and 2F1 to APCs that display the wild-type or the
modified peptides. Briefly, the murine TAP2-deficient RMA-S cells
transfected with the human HLA-A2 gene in an sc format
(HLA-A2.1/Db-2m sc) (RMA-S-HHD cells) were
loaded with the appropriate peptides, and the ability of the selected
Fab Abs to bind to peptide-loaded cells was monitored by FACS. First,
we confirmed a similar peptide-induced MHC stabilization of the TAP2
mutant RMA-S-HHD cells by the modified and wild-type gp100-derived
peptides. This was determined by analyzing the reactivity of the
conformation-specific anti-HLA Ab w6/32 and the anti-HLA-A2
mAb BB7.2 on cells loaded with modified or wild-type peptides
using FACS (Fig. 3).
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, A and B, the extent that
wild-type and modified HLA-A2-peptide complexes derived from gp100
epitopes G9-209 and G9-280, respectively, are displayed was identical
when probing with the conformation-specific monoclonal w6/32. Similar
results were observed when the display of the wild-type and modified
complexes for the two gp100-derived epitopes was monitored with BB7.2
specific for HLA-A2 (Fig. 3, C and D). These
results indicate that the extent of display, of modified and wild-type
gp100 epitopes, on peptide-stabilized RMAS-HHD cells is similar.
Similar results were observed when the modified and wild-type peptides
were loaded on the TAP+ JY APCs (data not
shown).
Next, we tested the binding of Fabs 1A9 and 2F1 to cells loaded with
the modified and wild-type peptides. We used the TAP-mutant RMAS-HHD
cells as described above and also JY APCs, which are EBV-transformed B
lymphoblast cells that express HLA-A2 and are
TAP+. Consequently, displaying the exogenous
peptide on these JY cells is facilitated by peptide exchange. Using
this strategy, we obtained a mixture of exogenously and endogenously
derived peptides presented on HLA-A2 that are displayed on the cell
surface. As shown in Fig. 4A,
Fab 1A9 binds to RMAS-HHD cells loaded with the G9-209M-modified
peptide, but not to cells loaded with the wild-type G9-209 peptide.
Similar results were observed when testing the binding of 1A9 to JY
cells loaded with modified or wild-type peptide; more specifically, 1A9
bound to cells loaded with the modified but not the wild-type peptide
(Fig. 4C). As shown in Fig. 3, the differential binding of
1A9 was not due to differences in the display of peptides (modified vs
wild-type) on the surface of the cells, because both were displayed to
the same extent. However, Fab 2F1 did not exhibit any differential
binding and was able to bind the wild-type as well as the modified
peptide on both RMAS-HHD (Fig. 4C) and JY cells (Fig. 4D).
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). As shown in Fig. 6, 1A9 binds to FM3D cells only when
pulsed with the modified G9-209M peptide, but not to cells pulsed with
the native G9-209 peptide (Fig. 6A). Moreover, 1A9 did not
bind to HLA-A2-negative KB3-1 cells pulsed with either peptide (Fig. 6B). In contrast to 1A9, Fab 2F1 binds to FM3D cells that
are pulsed with both the modified and the wild-type G9-280 peptide
(Fig. 6C), but not to peptide-pulsed KB3-1 cells (Fig. 6D). These results further corroborate the differential
binding of Fab 1A9 to the modified but not to the wild-type G9-209
epitope. Combined, these results demonstrate that altering the anchor
position 2 of G9-209 can dramatically influence the binding of TCR-like
Fabs as described in this study, which can be regarded as sensors to
the conformation of the Ag peptide in the context of the MHC
peptide-binding groove.
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To demonstrate that the TCR-like Fab Abs can be used as sensors
for monitoring conformational changes that cannot be detected by the
native TCR due to differences in the biophysical nature of these two
molecular entities, we tested the recognition of a CTL clone by scMHC
tetramers generated with wild-type and modified peptides. We have shown
previously that our scMHC tetramers, in which the human
2m gene is connected covalently to the three
extracellular domains of HLA-A2 using a 15-aa-long flexible linker, are
functional (27, 28). To determine whether the staining of
CTL clones can be influenced by the peptide present in the HLA
tetramers, we analyzed the staining pattern of two CTL clones derived
from patients who were vaccinated with the gp100-derived modified
epitopes G9-209M and G9-280V (32). As shown in Fig. 7, A–D, the
CD8+ CTL clone R6C12 (Fig. 7A),
specific for the G9-209M peptide, was stained intensively (80–90%)
with scHLA-A2 tetramers containing the wild-type as well as the
modified peptide (Fig. 7, B and C), but not with
tetramers containing the G9-280 epitope (Fig. 7D).
Similarly, the CD8+ CTL clone R1E2 (Fig. 7E) was stained intensively with scHLA-A2 tetramers
containing the wild-type or modified G9-280 epitope, but not the G9-209
epitope.
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Functional properties of anchor-specific TCR-like Abs
To further demonstrate the unique fine specificity of the
anchor-specific TCR-like Fab Ab 1A9, we tested its ability to interfere
with peptide-induced T cell activation of a gp100-specific T cell
clone. The TAP mutant human T2 cells, which express HLA-A2 molecules
when stabilized with an appropriate HLA-A2-restricted peptide, were
loaded with wild-type and modified peptide, and their ability to
activate the G9-209M-specific CD8+ CTL clone
R6C12 was tested by measuring the release of IFN-
to culture
supernatants (Fig. 8). As shown in Fig. 8A, T2 cells loaded with the wild-type or modified G9-209
peptide were able to activate the R6C12 cells to release IFN-,
whereas a control HLA-A2-restricted peptide derived from telomerase did
not induce CTL activation. When we tested the ability of the TCR-like
Fab Ab 1A9 to inhibit this peptide-specific CTL stimulation, we
observed that 1A9 was able to completely inhibit the stimulation of
R6C12 CTLs with T2 cells loaded with modified G9-209M peptide but not
stimulation induced by wild-type-loaded T2 cells (Fig. 8). The
inhibition of G9-209M-specific stimulation by Fab 1A9 was
dose-dependent with an apparent kilodalton of 50 nM, but no
inhibition was observed with G9-209 wild type-loaded T2 cells even when
high concentrations of Fab 1A9 (exceeding >15-fold the
Kd) were used. These results further
indicate the unique fine specificity of the TCR-like 1A9 Fab Ab and its
ability to be used as a very sensitive tool for studying
peptide-induced alterations in the conformation of the MHC-peptide
groove complex.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain, interacting with
the carboxyl end of the MHC 2 helix, and the TCR -chain
interacting with the carboxyl end of the MHC 1 helix. The CDR3
regions of the TCR and -chains interact with the center of the
peptide (P5-P7 depending on the peptide) (45, 46). Two characteristics about peptide binding to class I MHC are revealed by these structural informations. The first is that we have assumed that each amino acid in the peptide binds independently of one another to enhance or detract from the overall binding affinity. Considering this assumption, a popular algorithm was designed to predict peptide epitopes that bind well. This algorithm is based on the assumption that each residue binds independently (47). Although this algorithm predicts many good binding peptides from proteins of interest (48, 49, 50, 51), for unknown reasons it fails to predict accurately the results of single amino acid substitutions. The second characteristic of peptide binding explored in this study is that residues at the anchor positions do not affect the conformation of residues elsewhere in the peptide. If one assumes that each residue binds independently, homologous substitutions would be the best choice to amplify and activate T cells specific for the parental Ag (20, 21). The clear choice for modification is the anchor residues, because they point into the binding cleft and are restricted in space by the specificity pockets. Thus, the conformation flexibility of the peptide backbone should be limited, and any alterations in the structure caused by the anchor substitution would be expected to be local and small. In light of this consideration, peptides (termed altered peptide ligands) have been designed with increased affinity for class I MHC to enhance CTL stimulation (15, 20, 21, 23, 52, 53).
In this study, we have demonstrated that substitution in the anchor
residues can dramatically alter the overall conformation of the
peptide-MHC complex. Using a new molecular tool in the form of a
recombinant Ab endowed with the Ag-specific MHC-restricted specificity
of a TCR (TCR-like Ab), we have shown that the Ab can distinguish
between peptide-MHC complexes generated with a modified anchor residue.
TCR-like Abs are excellent molecules for use as probes for MHC-peptide
groove conformation. Peptides bound to MHC are deeply buried in the MHC
and are presented as extended mosaics of peptide residues intermingled
with MHC residues. Strikingly, no >100–300 Å2
of MHC class I-bound peptide faces outwards, and thus is available for
direct recognition. The MHC is thought to account for most of the
peptide-MHC complex surface presented to T cells. Abs recognizing
protein molecules engage 800 Å2 of their
ligand (54) so that Ab recognition of a peptide-MHC
complex (even if peptide-specific) would also have to be dominated by
the overall MHC conformation. Therefore, the surface area recognition
of a TCR-like Ab must encompass both the peptide and MHC. Indeed, as
shown in this study, such an Ab can be used to detect conformational
changes caused by modification of the peptide anchor position.
Further evidence supporting this came recently from a structural study demonstrating that class I MHC anchor substitution alters the conformation of TCR contacts (55). In this study, the binding to the class I MHC molecule HLA-A2 of a selection of known immunologically recognized peptide ligands from the tyrosine kinase family member HER-2/neu was examined.
Crystallographic analysis of these MHC complexes generated with modified peptides revealed that the TCR contact residues alter their positions depending on the identity of the anchor residue (55). The data presented show that substitutions in the center of a peptide bound to class I MHC may affect the positions of all of the residues within the peptide. In addition, small homologous substitutions in the anchor residues can dramatically alter the TCR-contacting residues. Clearly, the presence of substituted residues may alter the positions of nearly all of the other residues even when the substitution is a minor homologous substitution. The structural data in this study on the effect of modifying anchor positions is markedly enhanced by the data we have presented in this study demonstrating that the binding of a conformation-specific Ab with TCR-like properties is entirely dependent on the identity of a single anchor position.
An interesting question is how this can influence reactivity with T cells. T cells stimulated using altered peptide ligands are not necessarily the same population of T cells. Our study provides an explanation for the instances in which alteration of anchor position reduces or eliminates reactivity with T cells. The data presented by Sharma et al. (55) show that substitutions at the anchor positions can directly alter the conformation of the residues at the center of the peptide and conversely, that substitutions in the center can cause significant changes at the termini.
In the HLA-A2/G9-209 complex, position 2 is an HLA-A*0201 anchor residue that is not considered a TCR contact residue. However, the data presented in this study suggest that an HLA-A2 molecule containing the G9-209-2M peptide has a different conformation than an HLA-A2 molecule containing the wild-type parent G9-209 peptide. These conformational differences could be detected by the TCR-like G9-209-2M-specific Fab Ab 1A9. Importantly, these results indicate that modification of MHC binding anchor residues that do not have contact with the TCR can influence the overall conformation of the MHC peptide groove. The CTL clone chosen to test this in our study was not able to differentiate between the modified and the wild-type G9-209 peptide. However, in a recent study that analyzed the reactivity of TILs and T cell cloids derived from PBMC of patients vaccinated with G9-209-2M, changes in the fine specificity of the gp100 G9-209-reactive T cells clones were observed (24). It was found that immunization with the modified G9-209-2M peptide may have skewed the anti-G9-209 immune response in the vaccinated patients toward T cells with a reduced capacity to recognize tumor cells. In fact, the majority of peptide-reactive cloids did not recognize tumors. An interesting observation with respect to TCR recognition and differentiation between the wild-type and modified peptide was that several of the PBMC cloids were G9-209-2M-specific, and thus were not expected to be tumor-reactive because they do not recognize the parental G9-209 peptide. Other modifications in position 2 were also not recognized by these cloids.
This observation further strengthens our observation that modification of the anchor position 2 in G9-209, which is not considered a TCR contact residue, can influence the conformation of the MHC-peptide complex. The interesting findings by Clay et al. (24) suggest that T cells are capable of detecting these differences and that conformational changes induced by MHC binding anchor residues that do not contact the TCR could influence T cell reactivity. Further support for these observations came from studies using T cell clones that are specific for the same parental peptide in which altered peptide ligands resulted in differences in the T cell activation state among the different T cell clones.
In summary, the data presented in this study show that substitutions in anchor residues can dramatically alter the conformation of the peptide-MHC complex, and thus may have an effect on TCR-contacting residues. This may have important implications in cancer immunotherapy because most peptides studied are of low affinity; and therefore, modifications of anchor position are used to design peptides with increased affinity for class I MHC to enhance CTL stimulation.
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2 Address correspondence and reprint requests to Dr. Yoram Reiter, Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Room 333, Haifa 32000, Israel. E-mail address: reiter{at}tx.technion.ac.il
3 Abbreviations used in this paper: ER, endoplasmic reticulum; TIL, tumor-infiltrating lymphocyte; 2m, 2 microglobulin; sc, single chain; RT, room temperature.
Received for publication June 10, 2002. Accepted for publication August 15, 2002.
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  J. A. McWilliams, S. M. McGurran, S. W. Dow, J. E. Slansky, and R. M. Kedl A Modified Tyrosinase-Related Protein 2 Epitope Generates High-Affinity Tumor-Specific T Cells but Does Not Mediate Therapeutic Efficacy in an Intradermal Tumor Model J. Immunol., July 1, 2006; 177(1): 155 - 161. [Abstract] [Full Text] [PDF]
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O. Y. Borbulevych, T. K. Baxter, Z. Yu, N. P. Restifo, and B. M. Baker Increased Immunogenicity of an Anchor-Modified Tumor-Associated Antigen Is Due to the Enhanced Stability of the Peptide/MHC Complex: Implications for Vaccine Design J. Immunol., April 15, 2005; 174(8): 4812 - 4820. [Abstract] [Full Text] [PDF]
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K. Ida, S. Kawaguchi, Y. Sato, T. Tsukahara, Y. Nabeta, H. Sahara, H. Ikeda, T. Torigoe, S. Ichimiya, K. Kamiguchi, et al. Crisscross CTL Induction by SYT-SSX Junction Peptide and Its HLA-A*2402 Anchor Substitute J. Immunol., July 15, 2004; 173(2): 1436 - 1443. [Abstract] [Full Text] [PDF]
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W. E. Biddison, R. V. Turner, S. J. Gagnon, A. Lev, C. J. Cohen, and Y. Reiter Tax and M1 Peptide/HLA-A2-Specific Fabs and T Cell Receptors Recognize Nonidentical Structural Features on Peptide/HLA-A2 Complexes J. Immunol., September 15, 2003; 171(6): 3064 - 3074. [Abstract] [Full Text] [PDF]
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C. J. Cohen, O. Sarig, Y. Yamano, U. Tomaru, S. Jacobson, and Y. Reiter Direct Phenotypic Analysis of Human MHC Class I Antigen Presentation: Visualization, Quantitation, and In Situ Detection of Human Viral Epitopes Using Peptide-Specific, MHC-Restricted Human Recombinant Antibodies J. Immunol., April 15, 2003; 170(8): 4349 - 4361. [Abstract] [Full Text] [PDF]
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) or anchor-modified (G9-209M) (
) peptide and incubated with the
G9-209-specific HLA-A-restricted CTL clone R6C12 in the presence of
various concentrations of Fab 1A9 as indicated. T cell stimulation was
measured by the release of IFN-