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2-Microglobulin-Free HLA Class I Heavy Chain Epitope Mimicry by Monoclonal Antibody HC-10-Specific Peptide1
* Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy; and
Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263
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Abstract |
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2-microglobulin (2m). Furthermore, the HC-10 defined epitope appears to be involved in the pathogenesis of spondyloarthropathies, because HC-10 reduced their incidence in HLA-B27+2m°/MHC class II knockout mice. This study has characterized the determinant recognized by HC-10. Panning of a phage display peptide library with HC-10 resulted in isolation of the motif PxxWDR, which could be aligned with P57, W60, D61, and R62 of the first domain of the HLA-I HC allospecificities reactive with HC-10. The 55EGPEYWDR(N/E)T64 (p-1) is the shortest motif-bearing peptide that reacts with HC-10 and inhibits its binding to soluble HLA-B7 HC, irrespective of whether N (p-1a) or E (p-1b) is present at position 63. By contrast, HC-10 did not react with six additional peptides, each bearing motif amino acid substitutions present in HC-10-not-reactive HLA-I allospecificities. The p-1-derived Qp-1, synthesized with the additional conserved Q54, which displays the highest in vitro reactivity with HC-10, was the only one to induce in mice IgG resembling HC-10 in their fine specificity. Mapping of the HC-10-defined determinant suggests that the lack of mAb reactivity with 2m-associated HLA-I HC is caused by blocking by the peptide in the groove of 2m-associated HLA-I HC, though a role of HC conformational changes following its association with 2m cannot be excluded. This information contributes to our understanding of the molecular basis of the antigenic profiles of 2m-free and 2m-associated HLA-I HC and may serve to develop active specific immunotherapy of spondyloarthropathies. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-microglobulin (2m). It has long been known that association with 2m is required for transport and cell surface expression of the HLA class I (HLA-I) three molecular complex (1) and that the antigenic profile of 2m-associated HLA-I HC is markedly different from that of 2m-free HLA-I HC (2, 3). Therefore, with few exceptions (4), Abs reacting with 2m-free HLA-I HC do not react with the HLA-I three molecular complex (5, 6). mAb HC-10 is one of the few mouse mAb which react with 2m-free HLA-I HC (5, 7), but not with 2m-associated HLA-I HC. Whether this lack of reactivity reflects changes in the antigenic profile caused by the structural changes induced by association with 2m and/or steric hindrance because of close proximity of the antigenic determinant with the areas of the HC occupied by the peptide located in the HLA-I groove is not known. Like the mAb LA45 (8), L31 (9), and HCA2 (10), which also react with 2m-free HLA-I HC only, mAb HC-10 reacts with many, but not with all HLA-I HC (7, 11, 12). In addition, the epitope it recognizes appears to be involved in the pathogenesis of human-like spondyloarthropathies, because administration of mAb HC-10 reduces the incidence of the disease in B27+2m°/MHC class II knockout (A°) mice (13).
The determinant recognized by mAb HC-10 has been identified and characterized in the present study. This information contributes to our understanding of the molecular basis of the differences in the reactivity of mAb HC-10 with 2m-free and 2m-associated HC, as well as with classical and nonclassical HLA-I alleles and contributes to defining an immunogen to develop active specific immunotherapy of spondyloarthropathies.
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Materials and Methods |
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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 2m-associated and 2m-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 2m-free HLA-B and -C HC and on 2m-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 2m-associated HLA-A, B, C, E, and G HC (15, 16); the mAb TP25.99 (IgG1) which recognizes distinct determinants expressed on all 2m-associated HLA-A, B, C, and E HC and on all 2m-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 2m-free HLA-B HC were produced and characterized as previously described. The BALB/c mouse mAb 655 (IgG1) and its corresponding Ag, the human V-17 TCR-derived peptide GYSVSREKKES (p-V17), 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 x 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 x 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 -D-galactoside-isopropyl -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 x 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).
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Results |
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To identify the determinant recognized by mAb HC-10 on 2m-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).
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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).
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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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2m-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-V17 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-V17.
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17 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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2m-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-V17 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. 4A), suggesting that it resembles the mAb HC-10-specific binding site on 2m-free HC better than p-1a.
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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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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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2m-free HLA-I HC, and induce HC-specific Abs. Furthermore, the differential expression of this motif on HLA-I HC accounts for the differential reactivity of mAb HC-10 with classical and nonclassical HLA-I alleles, which had been previously defined by one-dimensional gel-isoelectric focusing (7, 11, 12) and immunofluorescence analysis (10). Alignment of phage-derived peptides showed that R62 is expressed by all HLA-I Ags reactive with mAb HC-10, indicating that this residue is crucial for the expression of the corresponding determinant. This conclusion is supported by the results obtained by Sernee et al. (24), who found by truncation experiments that amino acids surrounding residue 60 were essential for mAb HC-10 binding.
R62 was also found to be critical for the recognition of 2m-free HLA-I HC by mAb LA45 (8). At variance with the results obtained with mAb LA45 (8), N63 is not essential for the binding of mAb HC-10 to HLA-I HC as indicated by its reactivity with HLA-B*1302, B*1501, B*2705, and B*5201 alleles expressed on LBF (12), OLGA (12), JESTHOM (11), and KHAGHI (11) cell lines, respectively (Table III).
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Neither p-1a nor p-1b inhibited mAb HC-10 binding to 2m-free HC+ CEM. These results may reflect the higher association constant of mAb HC-10 for membrane-bound HC than for the synthetic peptides because the latter are in solution and/or because they mimic the determinant recognized by mAb HC-10 to a limited extent. Following addition of Q54, peptides p-1a and p-1b acquired the ability to inhibit the binding of mAb HC-10 to 2m-free HC+ CEM cells and induced IgG Abs with a specificity similar (or identical) to that of mAb HC-10 in BALB/c mice.
The immunogenicity of Qp-1b (54QEGPEYWDRET64) in BALB/c mice deserves some comments in view of its high degree of homology with 54QEGPEYWERET64 of H2-Dd Ags, which is one of the two MHC alleles expressed in BALB/c mice. The only difference between the latter sequence and the synthetic peptide at the level of motif amino acids (bold text) is replacement of the small polar D61 with the chemically similar E61. This difference is not likely to account for the immunogenicity of Qp-1 nor is it likely that this immunogenicity is caused by the adjuvant used for the immunization, because the 55EGPEYWDRET64p-1b mixed with this adjuvant did not elicit Abs at detectable levels in BALB/c mice. We favor the possibility that the immunogenicity of Qp-1b reflects the poor tolerogenic properties of this portion of 2m-free HLA-I HC, which is barely exposed on resting T cells (5). In view of the marked increase in the expression of 2m-free HLA-I HC expressing the mAb HC-10-defined epitope following T cell activation (5), it is tempting to speculate that following immune system activation, this epitope may trigger B and/or T cell-mediated autoimmune responses. If this interpretation is correct, the mAb HC-10-defined epitope may play a role in the induction of the auto-anti-2m-free HLA-I HC Abs found in the course of HIV infection (26) and provides a mechanism for the arthritogenic properties of the cytokeratin cross-reactive HLA-B27-derived peptide (spanning amino acids 60–72) (27), which partially overlaps the mAb HC-10-specific epitope. The close relationship between the arthritogenic and the mAb HC-10-specific epitopes of HLA-I HC may account for the ability of mAb HC-10 to prevent spontaneous inflammatory disease in HLA-B27 transgenic(B27+2m°) mice (13). These findings in conjunction with those about the immunogenicity of Qp-1 suggest that the latter may be a useful immunogen to induce mAb HC-10-like IgG Abs with the expectation that they will control inflammatory disease by inducing mAb HC-10-like IgG.
Mapping of the motif amino acids involved in the mAb HC-10-defined epitope in a three-dimensional model of HLA-I molecules has shown that R62 is spatially close to the peptide-binding pocket. This finding suggests that binding of mAb HC-10 to 2m-associated HLA-I HC may be inhibited by loading of the pocket with HLA-I Ag-binding peptides (6). Alternatively, the loading of the pocket with a high affinity peptide may induce conformational changes on residues critical for mAb HC-10 binding with the epitope loss, as envisaged in the mouse system with the anti-Kb mAb KU1 (28) and the anti-Ld mAb 64-3-7 (29). The latter two mAb and mAb HC-10 share a common ability to detect peptide free HC and recognize a similar region of the class I structure. mAb KU1 and 64-3-7 have their critical residues on a loop connecting a strand (under the bound peptide) with the 
1 helix (rising above the ligand binding site) (29), while mAb HC-10-defined peptide 55–64 maps to a region of an 1 domain, where the loop extends into helical conformation above the N terminus of the bound peptide (Fig. 8).
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2m with HLA-I HC does not interfere directly with mAb HC-10 binding because the corresponding determinant and the points of contact of 2m with the HC are spatially distant. This can partly explain the reported reactivity of mAb HC-10 with HC loosely but still associated to 2m (30). Another possibility is that mAb HC-10 binds an alternative conformation of the HC, not just a peptide-free, 2m-free form. This might explain why W60, which is hidden on mAb HC-10-reactive and nonreactive HLA-I (Fig. 7), seems to be crucial for the HC-10-defined epitope expression. It is reasonable to speculate that the peptide 55–64 including the mAb HC-10-defined motif, is in a more extended conformation on the 2m-free HC (31). Transfer of the mAb HC-10 epitope using mutagenesis to convert a nonreactive allele to a reactive one will be useful to address these points and to support HC-10-epitope definition in relation to conformational changes occurring when peptide and/or 2m bind or dissociate (29).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Franco Dammacco, Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine, University of Bari Medical School, Piazza G. Cesare 11, 70124, Bari, Italy. E-mail address: dimoclin{at}cimedoc.uniba.it
3 Abbreviations used in this paper: HC, H chain; 2m, 2-microglobulin; HLA-I, HLA class I; OPD, o-phenylenediamine.
Received for publication March 3, 2003. Accepted for publication June 6, 2003.
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References |
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2-microglobulin regulation of cell surface MHC class I conformation and expression. J. Immunol. 148:3723.[Abstract]
2-Microglobulin induces a conformational change in an MHC class I H chain that occurs intracellularly and is maintained at the cell surface. J. Immunol. 153:3533.[Abstract]
2-microglobulin-associated and 2-microglobulin-free HLA class I heavy chains. J. Immunol. 165:3275.2-microglobulin. J. Exp. Med. 171:1431.2-microglobulin from HLA class I heavy chains correlates with acquisition of epitopes in the cytoplasmic tail. J. Immunol. 154:5205.[Abstract]
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 M. A. Blanco-Gelaz, B. Suarez-Alvarez, S. Gonzalez, A. Lopez-Vazquez, J. Martinez-Borra, and C. Lopez-Larrea The amino acid at position 97 is involved in folding and surface expression of HLA-B27 Int. Immunol., January 1, 2006; 18(1): 211 - 220. [Abstract] [Full Text] [PDF]
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A. Facoetti, R. Nano, P. Zelini, P. Morbini, E. Benericetti, M. Ceroni, M. Campoli, and S. Ferrone Human Leukocyte Antigen and Antigen Processing Machinery Component Defects in Astrocytic Tumors Clin. Cancer Res., December 1, 2005; 11(23): 8304 - 8311. [Abstract] [Full Text] [PDF]
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L. Sibilio, A. Martayan, A. Setini, R. Fraioli, D. Fruci, J. Shabanowitz, D. F. Hunt, and P. Giacomini Impaired Assembly Results in the Accumulation of Multiple HLA-C Heavy Chain Folding Intermediates J. Immunol., November 15, 2005; 175(10): 6651 - 6658. [Abstract] [Full Text] [PDF]
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T. Gonen-Gross, H. Achdout, T. I. Arnon, R. Gazit, N. Stern, V. Horejsi, D. Goldman-Wohl, S. Yagel, and O. Mandelboim The CD85J/Leukocyte Inhibitory Receptor-1 Distinguishes between Conformed and {beta}2-Microglobulin-Free HLA-G Molecules J. Immunol., October 15, 2005; 175(8): 4866 - 4874. [Abstract] [Full Text] [PDF]
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M. Kloor, C. Becker, A. Benner, S. M. Woerner, J. Gebert, S. Ferrone, and M. von Knebel Doeberitz Immunoselective Pressure and Human Leukocyte Antigen Class I Antigen Machinery Defects in Microsatellite Unstable Colorectal Cancers Cancer Res., July 15, 2005; 65(14): 6418 - 6424. [Abstract] [Full Text] [PDF]
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G. V. Yamshchikov, D. W. Mullins, C.-C. Chang, T. Ogino, L. Thompson, J. Presley, H. Galavotti, W. Aquila, D. Deacon, W. Ross, et al. Sequential Immune Escape and Shifting of T Cell Responses in a Long-Term Survivor of Melanoma J. Immunol., June 1, 2005; 174(11): 6863 - 6871. [Abstract] [Full Text] [PDF]
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C.-C. Chang, M. Campoli, N. P. Restifo, X. Wang, and S. Ferrone Immune Selection of Hot-Spot {beta}2-Microglobulin Gene Mutations, HLA-A2 Allospecificity Loss, and Antigen-Processing Machinery Component Down-Regulation in Melanoma Cells Derived from Recurrent Metastases following Immunotherapy J. Immunol., February 1, 2005; 174(3): 1462 - 1471. [Abstract] [Full Text] [PDF]
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T. M. Tran, N. Satumtira, M. L. Dorris, E. May, A. Wang, E. Furuta, and J. D. Taurog HLA-B27 in Transgenic Rats Forms Disulfide-Linked Heavy Chain Oligomers and Multimers That Bind to the Chaperone BiP J. Immunol., April 15, 2004; 172(8): 5110 - 5119. [Abstract] [Full Text] [PDF]
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H. R. Turnquist, J. L. Petersen, S. E. Vargas, M. M. McIlhaney, E. Bedows, W. E. Mayer, A. G. Grandea III, L. Van Kaer, and J. C. Solheim The Ig-Like Domain of Tapasin Influences Intermolecular Interactions J. Immunol., March 1, 2004; 172(5): 2976 - 2984. [Abstract] [Full Text] [PDF]
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) 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.
), 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-V
) was included as a negative control. Results are expressed as percentage inhibition of binding compared with binding in the absence of inhibitor.
