β₂-Microglobulin-Free HLA Class I Heavy Chain Epitope Mimicry by Monoclonal Antibody HC-10-Specific Peptide

Animals

Female BALB/c mice (8- to 12-wk-old) were purchased from Charles River Breeding Laboratories (Milan, Italy).

Cells

The human T lymphoid cell line CEM (HLA-A1/31, -B8/40, -Cw7/9) was purchased from the American Type Culture Collection (Manassas, VA). Cultured human B lymphoid cells C1R-sB7 secreting large amounts of soluble β₂m-associated and β₂m-free HLA-B7 HC (14) were a kind gift from Dr. C. Grumet (Stanford University, Stanford, CA). Cells were grown in RPMI 1640 medium supplemented with 10% FCS (HyClone Laboratories, South Logan, UT) (complete medium) and 5 mM l-glutamine.

Conventional reagents, mAb, and peptides

Electrophoresis reagents were purchased from Bio-Rad (Hercules, CA). Unless otherwise specified, all chemicals were purchased from BDH Chemicals (Poole, U.K.).

The mAb HC-10 (IgG2a) which recognizes a determinant preferentially expressed on all β₂m-free HLA-B and -C HC and on β₂m-free HLA-A3, A10, A28, A29, A30, A31, A32, and A33 HC (Ref. 7 ; H. Grosse-Wilde, unpublished observation); the mAb W6/32 (IgG2a) which recognizes a monomorphic conformational determinant expressed on all β₂m-associated HLA-A, B, C, E, and G HC (15, 16); the mAb TP25.99 (IgG1) which recognizes distinct determinants expressed on all β₂m-associated HLA-A, B, C, and E HC and on all β₂m-free HLA-A, B, C HC except A2, A10, A29, A31, 32, 33, A68.1, A69, and B73 (4, 17); and the mAb L31 (9, 12) which recognizes a determinant preferentially expressed on β₂m-free HLA-B HC were produced and characterized as previously described. The BALB/c mouse mAb 655 (IgG1) and its corresponding Ag, the human Vβ-17 TCR-derived peptide GYSVSREKKES (p-Vβ17), were used as controls.

HRP-conjugated avidin and xenoantibodies to mouse IgG (Fc portion) were purchased from Jackson ImmunoResearch Laboratories (Avondale, PA). HRP-conjugated anti-M13 Abs were purchased from Pharmacia-LKB Biotech (Uppsala, Sweden).

mAb were purified from ascites by sequential precipitation with caprylic acid and ammonium sulfate (18) or by affinity chromatography on protein G (or protein A)-Sepharose (Pharmacia-LKB). Purity was assessed by SDS-PAGE as previously described (19). mAb concentration was determined in a bicinchoninic acid assay (Pierce, Rockford, IL). Purified mAb was coupled to biotin using the biotin-N-hydroxysuccinimide ester (Sigma-Aldrich, St. Louis, MO) (20) or to FITC (Sigma-Aldrich) (21) as previously described.

Soluble HLA-B7 HC were purified from spent medium of cultured C1R-sB7 cells as described elsewhere (22).

Peptides were synthesized at the Sigma “peptide synthesis service” (Sigma-Genosys, Cambridge, U.K.). Their quality was determined by analytical reverse phase chromatography and mass spectral analysis. Their purity was >80%.

Phage display peptide library

The Ph-12 Phage Display Peptide Library kit was purchased from New England Biolabs (Beverly, MA). This is a M13-filamentous phage display peptide library containing a repertoire of 1.2 × 10⁹ sequences of random 12 amino acid linear peptides fused at the N-terminal sequence of the M13 synthetic minor coat protein pIII (five for each virion), via the flexible linker GGGS, with an overall sequence PFYSHSX₁₂GGGSA.

Panning of peptide library with mAb HC-10

The panning of the peptide library with mAb HC-10 was performed according to the manufacturer’s instructions with the following modifications. Phage particles (2 × 10¹¹) were incubated with 30 μl of packed protein G- (first and third round) and protein A- (second and fourth round) Sepharose, previously coated with 30 μg of mAb HC-10-isotype-matched mAb W6/32. After a 1-h incubation at 4°C, unbound phage particles were recovered and incubated with 30 μl of packed protein A- (first and third round) and protein G- (second and fourth round) Sepharose previously coated with 30 μg of mAb HC-10. After an additional 1-h incubation at 4°C, beads were washed 10 times with 1 ml of TBS, containing 0.5% Tween 20, to remove unbound phage particles. Bound phage particles were eluted by addition of 500 μl of 0.2 M glycine buffer, pH 2.8. The eluate was rapidly neutralized with 75 μl of 1 M Tris-HCl, pH 9.1. Eluted phage particles from each round were amplified by infecting Escherichia coli ER2738 (in early growth phase) cultured in Luria-Bertani broth containing tetracycline (20 μg/ml; Sigma-Aldrich). E. coli cells were removed by centrifugation on the following day. Phage particles were purified twice by precipitation with a one-sixth volume of polyethylene glycol-NaCl (16.7%/3.3 M), resuspended in 200 μl of TBS buffer and used as input for the next round.

Phage enrichment (defined as percentage of eluted phage/input phage) at the end of panning was determined by spot titering eluted phage particles on Luria-Bertani plates containing 20 μg/ml tetracycline 5-bromo-4-chloro-3-indolyl β-d-galactoside-isopropyl β-d-thiogalactoside. Twenty single colonies were selected and amplified. Phage supernatants were tested in ELISA with mAb and used as a source for DNA purification.

ELISA screening of phage clones

Unless otherwise indicated, all incubation steps were performed at 25°C. PBS, PBS containing 0.05% Tween 20 (PBS-T20), and PBS containing 0.5% of BSA (PBS-BSA) were used as coating, washing, and blocking buffer, respectively.

Ninety-six-well microtiter plates (Falcon; BD Biosciences, Lincoln Park, NJ) were coated with mAb by incubating each well with 50 μl of a mAb solution (10 μg/ml) for 12 h at 4°C. After two washings and blockade of free protein-binding sites, 100 μl of supernatant containing amplified particles from each phage clone were added to wells and incubated for 2 h. Following four washings, wells were incubated for 1 h with an appropriate dilution of HRP-conjugated, affinity-purified goat anti-M13 Abs. Plates were then washed and developed by the addition of 100 μl of freshly prepared o-phenylenediamine (OPD) solution. Color reaction was stopped with a 100 μl/well solution of 2 N H₂SO₄. Absorbance was read at 492 nm with a Multiscan microplate reader (Benchmark; Bio-Rad). Background binding was determined by incubating phage supernatant in wells coated with PBS-BSA and with mAb HC-10 isotype-matched mAb W6/32.

Nucleotide sequence analysis of phage inserts

Phage DNA was purified from supernatant of clones reactive in ELISA by using a Qiaprep spin M13 Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions, and sequenced on an automatic DNA sequencer (30007 ABI Prism) at the MWG-BIOTECH DNA sequencing facility (Eberesberg, Germany) with a −96gIII promoter primer. Nucleotide sequences corresponding to peptide inserts were analyzed with the ExPASy DNA translate tool (http://expasy.hcuge.ch/) and deduced amino acid sequences were obtained. The motif was identified by alignment of peptide sequences from mAb HC-10-positive clones with the “Multalin Protein” tool at Pole Bio-Informatique Lyonnaise (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page = /NPSA/npsa_multalin.htm). A homology search was conducted in a SWISS-PROT database. The “ProteinInfo” tool at Prowl (http://prowl.rockefeller.edu/) was used to retrieve protein sequences bearing the motif.

Binding assay

The binding assay to test the reactivity of mAb or immune sera with synthetic peptides and purified HLA-B7 HC, and the inhibition assay to determine the ability of synthetic peptides to inhibit the binding of mAb to the peptide itself and to HLA-B7 HC were performed in 96-well microtiter plates (Falcon; BD Biosciences), as previously described (22).

Inhibition by a peptide of the binding of FITC-conjugated mAb HC-10 to cells was performed by mixing 40 μl of PBS-BSA solution containing 10-fold serial dilutions of inhibitor (starting concentration 100 μg/ml) with an equal volume of an appropriate dilution of FITC-labeled probe. Following a 1 h-incubation at 4°C, the mixture was added to target cells (2 × 10⁵) and incubation was prolonged for 1 h. Next, cells were washed once with 4 ml of PBS-BSA and their staining was analyzed with a cytofluorometer FACScan (BD Biosciences, San José, CA). Fluorescence data from 5000 viable cells were reported on a logarithmic scale. Controls included binding of FITC-labeled probe in the absence of inhibitor. Background binding was determined by staining cells with FITC-mAb HC-10 in the presence of a saturating amount of cold mAb.

BALB/c mice immunization

Three BALB/c mice were primed with an i.p. injection of 100 μg of purified peptide (or BSA) coupled to keyhole limpet hemocyanin in the presence of glutaraldehyde (23) and mixed with CFA (Invitrogen-Life Technologies, Carlsbad, CA). Mice were then boosted with 100 μg of the same immunogen in IFA on days 7 and 14. Serum was harvested on day 14 before the second booster and every week hereafter up to the eighth week. Sera drawn on day 35 displayed the highest binding titer with the corresponding immunogen and were used for the assays.

Immunochemical assays

Preparation of HLA-B7 HC-enriched supernatant, indirect immunoprecipitation with Abs, SDS-PAGE, and Western blotting were performed as described (22).

Analysis of motif amino acids on a three-dimensional model of the HLA-I molecule

Analysis of motif amino acids on a three-dimensional model of the HLA-I molecule was performed with the Swiss-PdbViewer at Glaxo Wellcome Experimental Research (http://www.expasy.ch/spdbv/mainpage.html).

Selection of phage-displayed peptides reactive with mAb HC-10

To identify the determinant recognized by mAb HC-10 on β₂m-free HLA-I HC, phage-displayed peptide clones were isolated by panning the random phage display peptide library with mAb HC-10. At each round, phage particles binding to isotypic and allotypic determinants of mAb HC-10 were removed by a preadsorption step on mAb HC-10 isotype-matched mAb W6/32. Similarly, protein A and protein G were alternatively used during panning to minimize the isolation of phage-displayed peptides reactive with these proteins.

Immunoscreening of 20 randomly selected colonies at the end of panning showed that they all reacted with mAb HC-10. The binding is specific because no reactivity with mAb W6/32 was detected. Eight mAb HC-10-reactive clones were selected for sequencing (Table I⇓).

Mapping of HC-10-specific epitope on HLA-I HC

Nucleotide sequence analysis of these clones identified seven distinct sequences in the peptides expressed by eight randomly selected independent clones. Alignment of the identified peptides is shown in Table I⇑. P, W, and D are present in six of the eight phage-derived peptides and R is present in all. A SWISSPROT-database search (http://prowl.rockefeller.edu/) showed that the motif PxxWDR (highlighted amino acids) could be aligned with amino acids P57, W60, D61, and R62 of the first domain of several HLA-I HC (Table II⇓), suggesting that this stretch is involved in their binding to mAb HC-10. Table II⇓ also presents the results of an alignment of the sequence of the HLA-B7 HC-derived peptide (spanning amino acids E55-T64) containing the motif, with the sequences of the same region from classical/nonclassical HLA-I alleles retrieved at the European Bioinformatics Institute database (http://www.ebi.ac.uk/imgt/hla/align.html). The motif amino acid substitutions identified were useful to define eight allele product groups. For each group a peptide was designed (p-1a/b to p-7).

Peptide binding and inhibition assays

The shortest motif-containing peptide giving a reliable reactivity with mAb HC-10 was p-1a/b (spanning from E55 to T64). Shorter peptides failed to bind mAb HC-10, whereas longer peptides, such as the p-1a/1b-derived Q54-T64 (Qp-1a/1b) synthesized with an additional (conserved) Q at position 54, displayed a higher reactivity with mAb HC-10 than p-1a/b. Representative results are shown in Fig. 1⇓.

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FIGURE 1.

Identification of the shortest motif (PXXWDR)-containing HLA-B7 HC-derived peptide binding to mAb HC-10. Fifty microliters of a PBS solution containing 5 μg/ml of peptides (indicated in each panel) were added to wells of a 96-well microtiter plate. Following a 12-h incubation at 4°C, three washings and blockade of the free protein-binding sites, 50 μl of 4-fold serial dilutions of mAb HC-10 (▪) were added to each well. After a 4-h incubation at 25°C and three washings, peptide-bound mAb was determined by sequential addition of an appropriate dilution of HRP-conjugated xenoantibodies to mouse IgG (Fc portion) and a freshly prepared OPD substrate solution. Color reaction was stopped with 100 μl of 2 N H₂SO₄. Absorbance was read at 492 nm in a Multiscan plate reader (Benchmark; Bio-Rad). Background binding was determined by replacing the experimental sample with PBS-BSA. Specific binding was determined by subtracting background binding from absorbance in the experimental well. mAb L31 (▴) and TP25.99 (•), which recognize determinants of HC distinct from that defined by mAb HC-10, were used as specificity controls.

Peptides p-2 to p-7 were synthesized with the same length as p-1a/1b and tested in a binding assay with mAb HC-10 to determine whether the resulting reactivity profile paralleled that previously obtained by mAb HC-10 with cell surface HLA-I allospecificities. Fig. 2⇓ shows that mAb HC-10 bound p-1b (and p-1a) in a dose-dependent manner. The binding is specific, because no reactivity was detected with equal amounts of mAb L31 and mAb TP25.99 which recognize determinants of β₂m-free HLA-I HC distinct from that defined by mAb HC-10 (4, 12). In contrast, mAb HC-10 did not react with peptides p-2, -3, -4, -5,-6, -7, and control p-Vβ17 bound to microtiter plates. Additional controls of the assay included the binding of mAb TP25.99 and L31 to HLA-B7 HC (sB7) and of mAb 655 to the corresponding p-Vβ17.

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FIGURE 2.

Effect of motif amino acid changes in peptides on their reactivity with mAb HC-10. Four-fold serial dilutions of a mAb HC-10 solution (▪) were added to wells coated with peptides, HLA-B7 HC (sB7), and the negative control p-Vβ17 (as indicated in each panel). After a 4-h incubation at 25°C and three washings, peptide-bound mAb was determined by sequential addition of HRP-conjugated xenoantibodies to mouse IgG (Fc portion) and OPD solution. Color reaction was stopped with 100 μl of 2 N H₂SO₄. Absorbance was read at 492 nm in a Multiscan plate reader (Benchmark; Bio-Rad). Binding of mAb L31 (▴) and TP25.99 (•) to a determinant of HC distinct from that seen by mAb HC-10, and of anti-p-Vβ17 mAb 655 (×), was included as the control. Motif amino acids are indicated in bold; amino acids different from those expressed in p-1b are underlined.

To rule out the possibility that mAb HC-10 did not react with p-2 to -7 because of their failure to bind to the plates, or due to changes in their conformation associated with their binding to plates, p-2 to -7 were tested for their ability to inhibit the binding of mAb HC-10 to wells coated with p-1a (Fig. 3⇓A) and p-1b (Fig. 3⇓B). Only p-1a and -1b displayed inhibitory activity. In both cases, the inhibition was dose-dependent and specific, because p-Vβ17 displayed no detectable inhibitory effect. In all subsequent experiments, p-1a/-1b and Qp-1a/-1b gave superposable results in terms of antigenicity and immunogenicity. Hence, only data related to p-1a and Qp-1a will be shown hereafter.

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FIGURE 3.

Effect of motif amino acid changes in peptides on their ability to inhibit mAb HC-10 binding to p-1a and p-1b. Fifty microliters of a PBS solution containing 4-fold serial dilutions of p-1a (•), p-1b (▴) p-2 (×), p-3 (⋄), p-4 (○), p-5 (▵), p-6 (+), and p-7 (∗) were mixed with an equal volume of an appropriate concentration of mAb HC-10 (3.12 μg/ml). After a 30-min incubation at 25°C, the mixture was added to wells coated with p-1a (A) and p-1b (B), and incubation was continued for an additional 4 h at 25°C. Wells were then washed, incubated with an appropriate dilution of HRP-conjugated xenoantibodies to mouse IgG (Fc portion), and developed by OPD solution. Color reaction was stopped with 100 μl of 2 N H₂SO₄. Absorbance was read at 492 nm in a Multiscan plate reader (Benchmark; Bio-Rad). Binding of mAb HC-10 in the presence of p-Vβ17 (□) was included as a negative control. Results are expressed as percentage inhibition of binding compared with binding in the absence of inhibitor.

To determine the extent of HC mimicry by p-1a and Qp-1a, their ability to inhibit mAb HC-10 binding to the corresponding soluble or cell surface Ag was evaluated in two sets of experiments. Fig. 4⇓ shows that p-1a inhibited the binding of mAb HC-10 to native (Fig. 4⇓A) and denatured (Fig. 4⇓B) β₂m-free HLA-B7 HC in a dose-dependent fashion. In both cases the inhibition is specific, because equal amounts of p-2 to p-7 and p-Vβ17 did not affect the binding of mAb HC-10 to HLA-B7 HC. In agreement with the results of the binding assay (Fig. 1⇑), Qp-1a displayed a greater inhibitory activity than p-1a (Fig. 4⇓A), suggesting that it resembles the mAb HC-10-specific binding site on β₂m-free HC better than p-1a.

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FIGURE 4.

Effect of motif amino acid changes in peptides on their ability to dose-dependently inhibit mAb HC-10 binding to insolubilized native (A) and denatured (B) HLA-B7 H chain. A, Fifty microliters of a PBS solution containing 4-fold serial dilutions of Qp-1a (•), p-1a (▴), p-2 (×), p-3 (⋄), p-4 (○), p-5 (▵), p-6 (+), and p-7 (∗), were mixed with an equal volume of an appropriate concentration of mAb HC-10 (3.12 μg/ml). After a 30-min incubation at 25°C, the mixture was added to wells coated with HLA-B7 HC and incubation was continued for an additional 4 h. Then, wells were washed, incubated with HRP-conjugated xenoantibodies to mouse IgG (Fc portion), and developed by OPD solution. Color reaction was stopped with 100 μl of 2 N H₂SO₄. Absorbance was read at 492 nm in a Multiscan plate reader (Benchmark; Bio-Rad). Binding of mAb HC-10 in the presence of p-Vβ17 (□) was included as a negative control. Results are expressed as percentage inhibition of binding compared with binding in the absence of inhibitor. B, Purified HLA-B7 HC (800 ng/lane) were run on a 12% SDS mini-gel, electrophoresed under reducing conditions, and transferred to a nitrocellulose filter (Bio-Rad) by applying a current of 250 mA for 20 min in a semidry blotting apparatus (Bio-Rad). After blocking of free protein-binding sites with PBS containing 0.5% Tween 20, the filter was incubated with an appropriate dilution of biotinylated mAb HC-10 previously mixed with p-1a at the indicated concentration. After a 2-h incubation at 25°C, Ag-Ab binding was detected by sequential addition of HRP-conjugated avidin and diaminobenzidine-based substrate solution. Binding of mAb HC-10 in the presence of p-4 was included as negative control.

This conclusion was corroborated in experiments that compared the ability of both peptides to inhibit the binding of mAb HC-10 to its corresponding epitope on membrane HC. As shown in Fig. 5⇓, only the peptide Qp-1a inhibited the staining of CEM cells by FITC-conjugated mAb HC-10 in a dose-dependent manner.

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FIGURE 5.

Differential inhibition by Qp-1a (A) and p-1a (B) of mAb HC-10 fluorescence staining of human T lymphoid CEM cells. Fifty microliters of an appropriate dilution of FITC-conjugated mAb HC-10 were mixed with an equal volume of PBS-BSA containing 1 (dotted line), 10 (dashed line), and 100 μg/ml (thin continuous line) of either Qp-1a (A) or p-1a (B). Following a 1 h-incubation at 4°C, the mixture was added to target cells (2 × 10⁵) and the incubation was prolonged for 1 h. Then, cells were washed and analyzed with a cytofluorometer. mAb HC-10 staining of cells in the presence of plain PBS-BSA (thick continuous line) was used as negative control. The unstained cell profile is indicated (dashed-dotted line).

Induction of anti-HLA-I HC Abs in mice immunized with peptides Qp1a and Qp1b

Additional experiments tested the ability of Qp-1a and Qp-1b to induce anti-HLA-I Abs. To this end, two groups of BALB/c mice (three mice/group) were immunized with the peptides. All the mice developed Abs reacting with the immunizing peptide. In addition, sera drawn on day 35, which have the highest titer with the immunizing peptide, reacted with purified HLA-B7 HC in Western blotting (representative results are shown in Fig. 6⇓, left panel). The reactivity is specific, because the immune sera did not react with BSA. The specificity of the assay was further indicated by the binding of mAb HC-10 and anti-BSA immune sera to HLA-B7 HC and to BSA, respectively, and by the lack of reactivity of the preimmune sera with both Ags. Blocking experiments showed that the Abs elicited by Qp-1a and Qp-1b specifically inhibited the binding of biotinylated mAb HC-10 to purified denatured HLA B7 HC in a dose-dependent fashion (representative results are shown in Fig. 6⇓, right panel).

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FIGURE 6.

Comparison of the fine specificity of sera elicited with Qp-1a and mAb HC-10. Left panel, Purified HLA-B7 HC (sB7) (lane 1) and BSA (control) (lane 2) (800 ng/lane) were run on a 12% SDS mini-gel, electrophoresed under reducing conditions, and transferred to a nitrocellulose filter that was then incubated for 3 h with mAb HC-10 (3 μg/ml) (A) and a 1/50 dilution of anti-Qp-1a (B) immune serum. Ag-Ab binding was detected by sequential addition of HRP-conjugated xenoantibodies to mouse IgG (Fc portion) and diaminobenzidine-based substrate solution. The binding of anti-BSA serum to HLA-B7 HC and to BSA was included as specificity control (C). Background binding was determined by replacing immune sera with preimmune sera (D). Right panel, Binding of biotinylated mAb HC-10 to HLA-I HC in the presence of anti-Qp-1a serum diluted 10 (lane 1), 50 (lane 2), and 250 (lane 3) times. Binding of mAb HC-10 in the absence of inhibitor (lane 4) and in the presence of a 1/10 dilution of anti-BSA serum (lane 5) was included as control.

It is noteworthy that peptides p-1a and p-1b are not immunogenic because they induced neither anti-peptide nor anti-HLA-I Abs in mice.

Mapping and surface accessibility analysis of HC-10-specific motif amino acids on a three-dimensional model of HLA-I molecule

Two established three-dimensional models of HLA-I molecules were retrieved from the Protein Data Bank at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/): one corresponds to the mAb HC-10-reactive HLA-Aw68 Ag (PDB accession number: 1HSB), bearing the motif PxxWDR (Fig. 7⇓, A and B), the other is the mAb HC-10 nonreactive HLA-A0201 Ag (PDB accession number: 1DUZ) with the motif amino acid R62 replaced by G62 (Fig. 7⇓, C and D). It can be seen that motif amino acids are closely related to the HLA-specific peptide binding pocket as they constitute a portion of its lateral wall. The position of the arbitrarily colored motif amino acids (Fig. 7⇓, A and C) indicates that the amino acid at position 62 comes into closer contact with the pocket. Furthermore, the access of R62 (with its side chain) to mAb HC-10 can be more readily hindered by the presence of the HLA-specific peptide in the pocket.

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FIGURE 7.

Schematic representation of the position of motif amino acids and their surface accessibility on a three-dimensional model of mAb HC-10-reactive HLA-Aw68 (Protein Data Bank accession number 1HSB) (A and B) and mAb HC-10-nonreactive HLA-A0201 (Protein Data Bank accession number: 1DUZ) (C and D). Motif amino acids were arbitrarily colored (P57, red; W60, black; D61, yellow; R62 or G62, green) (A and C) or colored according to their surface accessibility (from 100% surface exposed to 100% hidden: green, light green, light blue, and blue) (B and D). H and β₂m chains of HLA-I Ag are colored yellow and blue, respectively (A and C). Position of the HLA-specific peptide pocket is indicated (green dot).

Coloring of motif amino acids according to their surface accessibility (Fig. 7⇑, B and D) showed that in both HLA-Aw68 and HLA-A0201 molecules P57 is exposed (deep green) and W60 is completely hidden (deep blue), while D61 is more surface-accessible on HLA-A*0201. These results indicate different conformational changes between the two molecules at the level of the mAb HC-10-defined motif.