Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shields, M. J.
Right arrow Articles by Ribaudo, R. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shields, M. J.
Right arrow Articles by Ribaudo, R. K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 273, Issue 43, 28010-28018, October 23, 1998


The Effect of Human beta 2-Microglobulin on Major Histocompatibility Complex I Peptide Loading and the Engineering of a High Affinity Variant
IMPLICATIONS FOR PEPTIDE-BASED VACCINES*

Michael J. ShieldsDagger §, Ryuji Kubota, Wesley HodgsonDagger parallel , Steven Jacobson, William E. Biddison, and Randall K. RibaudoDagger **

From the Dagger  Laboratory of Immune Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892-1152 and the  Neuroimmunology Branch, NINDS, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The ability to directly load cell surface major histocompatibility complex (MHC) class I molecules with peptides provides a potentially powerful approach toward the development of vaccines to generate cell-mediated immunity. We demonstrate that exogenous beta 2-microglobulin (beta 2m) stabilizes human cell surface MHC I molecules and facilitates their loading with exogenous peptides. Additionally, using three-dimensional crystal structures and known interaction sites between MHC I heavy chains and beta 2m, we engineered variants of human beta 2m (hbeta 2m) with a single serine substitution at residue 55. This alteration was predicted to promote hydrophobic interactions at the MHC I heavy chain/beta 2m interface and displace an ordered water molecule. Compared with hbeta 2m, the serine to valine substitution at residue 55 had improved ability to bind to cell surface HLA-A1, HLA-A2, and HLA-A3 molecules, facilitate exogenous peptide loading, and promote recognition by peptide-specific T cells. The inclusion of hbeta 2m or higher affinity variants when pulsing cells with MHC-restricted peptides increases the efficiency of peptide loading 50-80-fold. Therefore, the inclusion of hbeta 2m in peptide-based vaccines may increase cell surface antigen densities above thresholds that allow recognition of peptide antigens by the immune system, particularly for cryptic, subdominant, or marginally antigenic peptides.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the field of vaccine development, it has been relatively simple to induce humoral responses to injected antigens. However, one of the major challenges in the treatment of tumors and viral infections is the generation of vaccines that stimulate cell-mediated immune responses to these pathogens. Specific cytolytic responses are generally mediated by CD8+ T cell recognition of antigenic peptides in the context of major histocompatibility complex (MHC)1 class I molecules. Recent advances in defining "supermotif" antigens capable of being presented by multiple MHC I alleles (1-4) and immunodominant epitopes (5-9) will undoubtedly have a significant impact on realizing these goals. However, beyond defining appropriate antigenic peptides, a second challenge lies in establishing effective methods with which to deliver these antigens to the MHC I loading pathway. Typically, MHC I molecules acquire peptides generated by the degradation of endogenous proteins by the proteasome. These peptides are transported into the endoplasmic reticulum, where they are bound by MHC I·beta 2-microglobulin (beta 2m) complexes and finally transit to the cell surface (10-12). Although this pathway is the dominant means of loading MHC I molecules, other methods of delivering antigenic peptides to MHC I have been described including direct incorporation of DNA into cells (13-16), phagocytosis-dependent representation of antigens (17, 18), and infection by bacterial (19) or viral vectors (20, 21). All of these approaches attempt to introduce peptide into the endogenous loading pathway. Alternatively, direct cell surface loading of MHC I molecules in vitro has also been demonstrated but is inefficient in the absence of serum (22, 23). Thus, peptide antigens can be bound to MHC I molecules, but they must either be introduced into the endogenous pathway or be loaded exogenously.

Some active and approved clinical trials involve the direct injection of free peptide (usually in an oil-based adjuvant) into patients to induce cell-mediated immunity (e.g. Protocol ID numbers NCI-95-C-0145D, NCI-T95-0031N, NCI-95-C-0143B, NCI-T95-0039N, MSKCC-95052, NCI-H95-0769, LAC-USC-10M954, NCI-T95-0069D, NCI-97-C-0141, NCI-T96-0078, NCI-98-C-0023, NCI-98-C-0022, 94-C-0159).2 The most likely mechanism for the formation of peptide-MHC I complexes in these cases is the exogenous binding of free peptide to cell surface MHC I molecules. Successful exogenous loading requires that added peptides bind to cell surface MHC I molecules by exchange of their endogenously derived peptides. Since MHC I molecules that have lost their endogenously loaded peptides are inherently unstable (24), efficient loading of exogenous peptide presents a significant challenge. We, as well as others, have demonstrated that empty MHC I molecules can be stabilized with exogenous human beta 2m (hbeta 2m) (23, 25, 26). Thus, the generation of higher affinity variants of beta 2m could both quantitatively and qualitatively affect the efficiency of exogenous peptide loading by increasing both the amount of peptide loaded as well as the length of time that the peptide remains bound. These qualitative and quantitative effects may allow the immune system to generate cellular responses to lower affinity peptides. Responses to lower affinity antigens may in turn lead to recognition and destruction of neoplastic or virally infected cells that would otherwise be below the threshold of recognition by the immune system.

Site-directed mutagenesis of MHC I heavy chains has identified residues that affect peptide association, T cell recognition, and stability with beta 2m (27-29). Additionally, a great deal of work has been done identifying, analyzing, and modifying peptides bound to MHC molecules. In contrast, however, much less work has been done engineering and comparing different beta 2m molecules, with a few notable exceptions comparing murine with human beta 2m and mutagenizing hbeta 2m (26, 30-35). Further, comparisons of hbeta 2m and murine beta 2m (mbeta 2m) reveal that hbeta 2m binds with higher affinity to murine MHC I heavy chains than mbeta 2m (31), and we have identified a region of hbeta 2m (the S5 strand) responsible in part for this effect (26). Using this information and the available three-dimensional crystal structures of MHC I molecules, we engineered a variant of hbeta 2m to create an ionic bond that stabilized interactions with the murine MHC I heavy chain H-2Dd to a greater extent than wild-type hbeta 2m (26).

We have now extended this work to human MHC I molecules. In this report we have utilized four independent means to compare variant hbeta 2m molecules: 1) an assay in which exogenous beta 2m and peptide stabilize cell surface MHC molecules that have been stripped of their endogenous beta 2m and peptide (36, 37); 2) an inhibition assay we have developed that directly compares the ability of different beta 2m species to bind to cell surface MHC molecules; 3) direct peptide binding to MHC I molecules using a FITC-labeled peptide; and 4) a cytotoxic T lymphocyte (CTL) lysis assay, which indirectly measures the loading of antigenic peptides onto target cells based on the lysis of those target cells by CTL clones. We have engineered a higher affinity variant of hbeta 2m that, by the above criteria, stabilizes human MHC I molecules to a greater extent than wild-type hbeta 2m, enhances peptide binding, and enhances the ability of CTL to recognize peptide-loaded target cells. Further, this stabilizing activity was observed with all three HLA alleles examined: HLA-A1, HLA-A2, and HLA-A3.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Site-directed Mutagenesis

hbeta 2m cDNA in Bluescript SK (Stratagene, La Jolla, CA) was mutated using the ExSite mutagenesis system (Stratagene) according to the manufacturer's protocol and subcloned into the bacterial expression vector pET-21d(+) (Novagen, Madison, WI) using engineered NcoI and BamHI sites at the 5'- and 3'-end of the mature protein sequence, respectively. The D53 variants were described previously (26). The oligonucleotides used for the S55 mutagenesis were as follows: sense S55I, 5'-ATT TTC AGC AAG GAC TGG TCT TTC-3'; sense S55V, 5'-GTG TTC AGC AAG GAC TGG TCT TTC-3'; common antisense, 5'-TAA GTC TGA ATG CTC CAC TTT TTC-3'.

The oligonucleotides used for the insertion of the 9E10 epitope (amino acid sequence EQKLISEEDLN) at the amino terminus of wild-type beta 2m to create the myc-beta 2m were as follows: sense myc, 5'-TCC GAG GAG GAC CTG AAC ATC CAG CGT ACT CCA AAG ATT CAG G-3'; Antisense myc, 5'-AAT AAG CTT CTG CTC CAT GGC CTC GAG GCC AGA AAG AGA GAG-3'. Mutagenized or added regions are underlined. Constructs were confirmed by sequence analysis using standard techniques.

Synthesis and Purification of Recombinant beta 2m

Recombinant beta 2m expression and purification has been described previously (26). Briefly, beta 2m constructs in pET-21d(+) were transformed into the BL21(DE3) strain of Escherichia coli. At an A600 of 0.6, cultures were induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 4 h, and inclusion bodies were isolated by centrifugation after sonication of bacteria in 200 mM Tris, 2 mM EDTA, 10% Triton X-100, pH 7.6, and washing in 200 mM Tris, 2 mM EDTA, pH 7.6. Inclusion bodies were solubilized in 6 M guanidine HCl containing 0.3 M dithiothreitol, 100 mM Tris, pH 8.0, and a mixture of protease inhibitors (5 µg/ml leupeptin, 0.5 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 1% aprotinin). Following overnight dialysis in 6 M guanidine, pH 2.0, recombinant protein was refolded over 72 h in 0.4 M arginine, 5 mM oxidized glutathione, 100 mM Tris, 2 mM EDTA at 10 °C. Following refolding, preparations were dialyzed exhaustively against 0.4 M arginine, 100 mM Tris, 2 mM EDTA, pH 8.0, and then PBS at 4 °C. Preparations were purified as a single peak by preparative fast protein liquid chromatography on a Superdex 75pg gel filtration column (Amersham Pharmacia Biotech), concentrated using Centriprep-3 concentrating units (Amicon, Beverly, MA), and sterile-filtered, and concentrations were calculated based on A280 readings. Recombinant beta 2m was judged to be >= 95% pure based on analysis by SDS-polyacrylamide gel electrophoresis, and analytical fast protein liquid chromatography. Independent preparations have been compared for specific activity and shown to be identical at a molar level with no loss in activity when stored at -70 °C for >6 months and at 4 °C for >2 months. Recombinant hbeta 2m was also shown to be identical in activity to native hbeta 2m purified from urine (Sigma) (38).

Cell Lines and Abs

Hmy2.C1R cells (39) were stably transfected with HLA-A1, -A2, and -A3 as described previously (40, 41). HLA-A2/HTLV-1 tax 11-19 peptide-specific CTL clone N1218 and HLA-A3/influenza NP 265-273 peptide clone 2G12 were isolated and restimulated as described previously (42). All mAbs were used as culture supernatants grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 20 mM HEPES, 2 mM L-glutamine, 1% nonessential amino acids, 1% penicillin/streptomycin, and 0.04 mg/ml of Gentamicin sulfate (complete Dulbecco's modified Eagle's medium). GAP.A3 (HLA-A3-specific) and BB7.5 (pan-HLA-ABC-specific) hybridomas were obtained from the American Type Culture Collection (Rockville, MD). The myc-specific 9E10 hybridoma has been previously described (43). Unless otherwise noted, all solutions used for cell growth were obtained from Biofluids (Rockville, MD).

Peptides

Peptides were as follows: the HLA-A1 binding ornithine decarboxylase 309-317 (OD 309), SSEQTFMYY; the HLA-A2-binding HTLV-1 TAX 11-19, LLFGYPVYV; HIV gag 77-85, SLYNTVATL; the FITC-conjugated HIV RT 195-207 peptide, ILK*EPVHGV (the FITC moiety is conjugated to the lysine residue); the HLA-A3-binding pn2a.A3, KLYEKVYTYK; and influenza NP 265-273, ILRGSVAHK (44-49). The peptides were purchased from Bachem (Torrance, CA), provided by Drs. Jack Bennink and Jonathan Yewdell, or provided by John E. Coligan (NIAID, NIH). All peptides were purified by reverse phase HPLC and were >95% pure as determined by analytical HPLC and mass spectrometry.

Cytotoxic T Lymphocyte Lysis Assays

Cytolytic activity was assessed using a modified Europium release assay (50) as follows. Hmy2.C1R cells transfected with HLA-A2 or HLA-A3 were resuspended to 4 × 106 cells/ml in complete Dulbecco's modified Eagle's medium supplemented with 20 µM BATD (which forms a fluorescent chelate with Europium; Wallac, Gaithersburg, MD) and incubated at 37 °C for 30-60 min. Cells were resuspended in 10 ml of serum-free (SF) CTL medium (Iscove's modified Dulbecco's medium (IMDM) supplemented with 5 mg/ml bovine serum albumin (BSA; Sigma), 2 mM L-glutamine, 1.25 mM sulfinpyrazone (Sigma), and 1% penicillin/streptomycin), centrifuged, and washed once more with SF CTL medium. Cells were then pulsed with peptide with or without beta 2m in SF CTL medium for 60-90 min at 37 °C. Cells were washed twice in SF CTL medium, resuspended in CTL medium (5% fetal calf serum in lieu of BSA), and combined at the designated effector:target ratio with CTL clones in round bottom microtiter plates. Plates were gently centrifuged at 100 × g for 2 min and then incubated at 37 °C for 2 h. Finally, plates were centrifuged at 300 × g, and 20 µl/well was transferred to 200 µl of 0.3 M acetic acid, 60 mM sodium acetate, 7.5 µg/ml Europium (Aldrich, Milwaukee, WI), and the plate was read on a Wallac 1234 DELFIA fluorometer. The percentage of specific lysis was calculated with the following equation: 100 × ((experimental - blank) - (spontaneous - blank))/((maximum - blank) - (spontaneous - blank)). Triplicate samples were used, and error bars indicate S.D. values.

FITC-conjugated Peptide Binding to Cell Surface MHC I Molecules

Hmy2.C1R-A2 cells were resuspended in SF IMDM (IMDM supplemented with 2.5 mg/ml BSA, 20 mM HEPES, 2 mM L-glutamine, 1% nonessential amino acids, 1% penicillin/streptomycin, and 0.04 mg/ml Gentamicin sulfate) with the indicated concentrations of peptide with or without recombinant beta 2m and incubated at 37 °C for 17 h. Cells were washed twice with PBS plus 2 mg/ml BSA (PBSA) and resuspended in PBSA followed by flow cytometric analysis on a FACS analyzer (Becton Dickinson, Mountain View, CA). 8000-20,000 live (propidium iodide-excluding) events were collected per sample, and values are expressed as mean fluorescence intensity. ED50 values were calculated using a sigmoid logistic fit.

Cell Surface MHC Stabilization of Acid-stripped Cells

The MHC stabilization was done essentially as described previously (36, 37, 51) with minor modifications. Briefly, Hmy2.C1R-A1, -A2, and -A3 cells were washed twice with PBS, resuspended in 0.13 M citric acid, 66 mM Na2HPO4, pH 2.9 (pH 3.2 for A2 cells), for 90 s at 4 °C, washed with two 50-ml changes of IMDM, and resuspended in SF IMDM. 105 cells/well were added to a 96-well microtiter plate containing hybridoma supernatants, peptide, and beta 2m dilutions in a total volume of 150 µl. After a 4-h incubation at 23 °C, cells were washed twice with PBS, 2 mg/ml BSA, 0.02% NaN3 (FACS buffer) and stained with FITC-conjugated goat anti-mouse IgG (H + L) F(ab')2 fragment (Cappel/Organon Teknika, Durham, NC) for 1 h at 4 °C. Cells were washed twice with FACS buffer and fixed in 1% formaldehyde in PBS followed by flow cytometric analysis on a FACScan II (Becton Dickinson, Mountain View, CA). Values are expressed as mean fluorescence intensity.

myc-hbeta 2m Binding and Inhibition Assays

myc-beta 2m Binding Assay-- Hmy2.C1R transfectant cells at 2.5 × 105 cells/tube in a 500-µl volume were incubated at 37 °C for 16 h in SF IMDM with different concentrations of myc-beta 2m with or without peptide. Cells were washed three times in plain IMDM followed by incubation with 9E10 (anti-myc) hybridoma supernatant at 4 °C for 1 h. After washing with IMDM, cells were stained for 1 h with FITC anti-mouse IgG at 4 °C for 1 h. Cells were washed a final time in FACS buffer, and live cells (propidium iodide-excluding) were analyzed by flow cytometry.

myc-beta 2m Inhibition Assay-- The inhibition assay is identical to the binding assay with the following modifications: 2.5 µM myc-beta 2m was used in all cases, and different concentrations of non-myc-labeled recombinant beta 2m were included to inhibit myc-beta 2m binding to cell surface MHC molecules. The percentage of inhibition was calculated by the following equation: (1 - ((experimental - background)/(no inhibitor - background))) × 100. 10,000-20,000 gated events/sample were counted, and all experiments were repeated at least twice. ID50 values were calculated using a sigmoid logistic fit.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

beta 2m Enhances CTL Recognition of Target Cells and Exogenous Peptide Loading-- The effectiveness of beta 2m in facilitating exogenous peptide loading of MHC I molecules was measured using a CTL lysis assay. Target cell lysis not only correlates with the loading of a specific peptide antigen, but it also demonstrates that the peptide is bound in an immunologically relevant manner. Hmy2.C1R-A2 target cells (a human lymphoblastoid cell essentially null for HLA molecules except for the transfected HLA-A2.1) (40, 41) were pulsed with a suboptimal concentration of HTLV-1 TAX peptide for 90 min in SF CTL medium in the absence or presence of increasing concentrations of purified, recombinant hbeta 2m and then used as targets in a conventional lysis assay. The presence of hbeta 2m dramatically increased the specific lysis by the TAX-specific CTL clone in a dose-dependent fashion (Fig. 1). Lysis at the maximal amount of hbeta 2m in the presence of an irrelevant A2-binding peptide was at background levels. Using this suboptimal concentration of peptide, there was 20% lysis in the absence of beta 2m. The addition of 8 µM beta 2m increased the lysis to the maximum observed at this effector:target ratio. In the absence of beta 2m, a 50-100-fold higher concentration of peptide would be required to achieve comparable levels of lysis.3


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 1.   Enhancement of CTL recognition of target cells by human beta 2m. Hmy2.C1R-A2 target cells were pulsed with BATD (Europium chelator) for 30 min and washed in serum-free CTL medium followed by incubation with a suboptimal dose of TAX peptide (9.3 pM) with the indicated concentrations of hbeta 2m for 90 min. Target cells were washed and then combined with the TAX-specific HLA-A2-restricted CTL clone N1218 at an effector:target ratio of 4:1 for 2 h. 20 µl of supernatant was harvested, added to 200 µl of Europium solution, and read on a Wallac 1234 DELFIA fluorometer. The specific lysis of target cells pulsed with the irrelevant A2-binding HIV gag 77-85 peptide at 1 nM in the presence of 8 µM hbeta 2m was 29 ± 6%. All incubations were done at 37 °C.

To directly demonstrate the effectiveness of hbeta 2m to enhance peptide loading of MHC I molecules, we measured the binding of a FITC-labeled, HLA-A2-restricted peptide (HIV RT 195-207) to Hmy2.C1R-A2 cells. As shown in Fig. 2a, the presence of hbeta 2m shifted the peptide titration curve approximately 50-fold compared with peptide loading in the absence of hbeta 2m, consistent with the magnitude of shift observed for the augmentation of CTL lysis.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 2.   Human beta 2m facilitates peptide binding to cell surface HLA-A2. a, Hmy2.C1R-A2 cells were centrifuged and resuspended in serum-free IMDM (SF IMDM) with the indicated concentrations of the FITC-conjugated HLA-A2-restricted HIV RT 195-207 peptide with (black-square) or without (black-diamond ) 10 µM hbeta 2m for 17 h at 37 °C. Samples were washed twice with 2 mg/ml BSA in PBS, resuspended, and analyzed by flow cytometry. All values are expressed as mean fluorescence intensity. b, individual histograms of cells incubated with 333 ng/ml FITC-peptide in SF IMDM with or without 10 µM hbeta 2m.

The S55V Mutation Enhances MHC Molecule Stability on the Cell Surface-- Based on previous work, the region of hbeta 2m including the S5 strand was found to play an important role in the higher affinity of hbeta 2m for murine MHC I heavy chains (26) and was the focus of our structural search for mutations to increase the affinity of hbeta 2m. Using the HLA-A2 crystal structure (52),4 we identified a hydrophilic serine residue at position 55 of hbeta 2m that was buried at the hbeta 2m/heavy chain interface and was situated directly adjacent to an ordered water molecule. This residue was substituted with hydrophobic residues of increasing mass (valine and isoleucine) in order to promote hydrophobic interactions and exclude the ordered water. The valine side chain was chosen, since it is approximately the volume of a serine side chain hydrogen-bonded to a water molecule. Serine 55 was also changed to isoleucine, since it was unclear if and how the hydrophobic residues would pack in relation to other side chains at the hbeta 2m/heavy chain interface.

We initially used an HLA stabilization assay (36, 37) to screen the beta 2m variants. Since there can be significant allele-specific differences in the ability of hbeta 2m point mutants to stabilize murine heavy chains3 (26) a number of HLA alleles, HLA-A1, HLA-A2, and HLA-A3, were analyzed in order to determine the generality of any observed effects.

Fig. 3 demonstrates the ability of the S55 substituted beta 2ms to stabilize HLA-A1 (Fig. 3a), HLA-A2 (Fig. 3b), and HLA-A3 (Fig. 3, c and d) in the presence of a specific binding peptide and an appropriate HLA-specific antibody. The S55V variant (Fig. 3, ×) stabilized HLA-A1 and HLA-A3 approximately 2- and 3-fold better, respectively, than wild-type hbeta 2m (diamonds) at a molar level, and effects on HLA-A2 stabilization by S55V were only slightly better than those observed with wild-type hbeta 2m. The effects of S55I (triangles) varied depending on the allele: better along the upper end of the titration curve with HLA-A1, worse with HLA-A2, and marginally better with HLA-A3. We have previously demonstrated that a single mutation at residue 53 (D53N or D53V) of hbeta 2m decreased the affinity of its interaction with murine heavy chains, presumably by disruption of a conserved, coordinated ionic bond (26). As was observed with murine heavy chains, both Asp53 mutants (circles and stars) were deficient relative to wild-type hbeta 2m at stabilizing all three human heavy chain alleles.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 3.   The stabilization of cell surface HLA-A1, -A2, and -A3 by mutant beta 2m. Cells stripped of their endogenous beta 2m and peptide were combined with the indicated concentrations of mutant or wild-type hbeta 2m with peptide and a beta 2m/peptide-dependent mAb for 4 h at 23 °C. black-diamond , wild-type hbeta 2m; black-triangle, S55I; ×, S55V; *, D53N; bullet , D53V. Cells were washed, stained with FITC-labeled anti-murine IgG, and then analyzed by flow cytometry. All values are expressed as mean fluorescence intensity. HLA-A1-transfected Hmy2.C1R cells (a) were combined with BB7.5 mAb and 10 µg/ml A1-binding OD 309 peptide. HLA-A2-transfected Hmy2.C1R cells (b) were combined with BB7.5 mAb and 2.5 µg/ml A2-binding HIV gag peptide. HLA-A3 transfected Hmy2.C1R cells were combined with BB7.5 (c) or GAP.A3 (d) mAb and 1.25 µg/ml A3-binding pn2a.A3 peptide. Panel d displays error bars corresponding to the S.D. values of three independent data points.

We observed a decline in the stabilization of all three MHC I alleles at the highest concentrations of beta 2m tested when using mAb BB7.5 (Fig. 3, a-c). The choice of this mAb for the stabilization experiments in Fig. 3 was determined by the mAb's dependence on both beta 2m and peptide for the formation of the epitope as well as the fact that it was reactive with all three alleles studied. Similar high dose inhibition was also observed even when freshly isolated cells were incubated on ice in the presence of BB7.5,3 suggesting that this effect was due to inhibition of BB7.5 binding by free beta 2m in solution, consistent with beta 2m forming part of the combinatorial BB7.5 epitope (53). Using an alternative, HLA-A3-specific mAb to compare wild-type hbeta 2m and S55V, no inhibition was observed (Fig. 3d). Further, the 2.5-fold molar increase in the ability of the S55V mutant to stabilize HLA-A3 was consistent with the 3-fold increase when using BB7.5 (Fig. 3c). Interestingly, the pan-HLA-ABC-specific mAb W6/32 could not be used due to its inability to bind MHC complexes containing recombinant beta 2m, which have an additional methionine at the amino terminus (38).

The Peptide Dependence of hbeta 2m Binding and Its Augmentation by S55V-- The antibodies used in Fig. 3 were selected due to their dependence on both beta 2m and peptide in order to detect "complete" molecules, i.e. heavy chain·beta 2m·peptide natively folded trimeric complexes. Since this binding assay required the presence of an antibody in addition to beta 2m and peptide (37), there was formally the possibility that the antibodies exerted an effect that was specific for a particular beta 2m mutant such as S55V. Due to previously reported concerns regarding the potential contribution of the antibodies to the stabilization of cell surface MHC I complexes (54), we extended the stabilization studies by developing a binding inhibition assay that directly measured the relative abilities of beta 2ms to bind to MHC I molecules. To do this we engineered an epitope tag (myc) onto the amino terminus of hbeta 2m so that its binding could be directly measured with the anti-myc mAb 9E10. The addition of this tag to the amino terminus was preferable to biotinylation, iodination, or other labeling procedures that could variably alter residues involved in heavy chain/beta 2m interactions and therefore require further purification (55).

Hmy2.C1R cells transfected with HLA-A1, -A2, or -A3 alleles were incubated overnight in SF medium with increasing concentrations of myc-beta 2m (Fig. 4). In the presence of an appropriate peptide, there was concentration-dependent myc-beta 2m binding for all alleles studied. However, when cells were incubated with myc-beta 2m in the absence of peptide, very little myc-beta 2m binding was observed. It should be noted that these experiments were conducted for 16 h, whereas comparable or even greater binding of human or murine beta 2m to murine MHC I molecules occurred in only 2 h even in the absence of peptide.5 This suggests some fundamental differences in the stability and behavior of murine and human MHC I molecules.


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 4.   The binding of myc-beta 2m to cell surface HLA-A1, -A2, and -A3. Hmy2.C1R-A1 (a), -A2 (b), and -A3 (c) cells were incubated with the indicated concentrations of myc-beta 2m with (black-square) or without (black-diamond ) 20 µg/ml peptide in serum-free IMDM for 16 h at 37 °C. After washing away free myc-beta 2m, cells were stained with 9E10 (anti-myc) culture supernatant, stained with FITC-labeled anti-murine IgG, and then analyzed by flow cytometry. All values are expressed as mean fluorescence intensity.

Having established conditions in which myc-beta 2m binding could be demonstrated, the relative abilities of wild-type hbeta 2m and S55V to inhibit the binding of myc-beta 2m to HLA molecules were next compared (Fig. 5). Compared with wild-type hbeta 2m, the S55V mutant inhibited myc-beta 2m binding about 2-fold better at a molar level for HLA-A1, -A2, and -A3. These results are consistent with a higher relative affinity of the S55V mutant compared with wild-type hbeta 2m for HLA-A1, -A2, and -A3.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 5.   Inhibition of myc-beta 2m binding by S55V and hbeta 2m to cell surface HLA-A1, -A2, and -A3. Hmy2.C1R-A1 (a), -A2 (b), and -A3 (c) cells were incubated with 2.5 µM myc-beta 2m and 20 µg/ml peptide in serum-free IMDM with the indicated concentrations of inhibitor wild-type hbeta 2m (black-diamond ) or S55V (black-square) for 16 h at 37 °C. The OD 309 peptide was used for HLA-A1, the HIV gag peptide for HLA-A2, and the pn2a.A3 peptide for HLA-A3. After washing away free beta 2m, cells were stained with 9E10 (anti-myc) culture supernatant, stained with FITC-labeled anti-murine IgG, and then analyzed by flow cytometry. All values are expressed as mean fluorescence intensity. The 50% inhibitory dose values were calculated for each curve and listed with their respective graphs.

The S55V Mutant Enhances Peptide Loading and CTL Recognition-- Having established the higher relative affinity of the S55V mutant for HLA-A1, -A2, and -A3, we next studied its ability to enhance the loading of peptide onto Hmy2.C1R-A2 cells directly with the FITC-labeled HIV RT peptide. In the presence of a limiting concentration of FITC-peptide (100 ng/ml; see Fig. 2a), hbeta 2m and S55V were titered onto cells to measure their ability to enhance exogenous peptide loading (Fig. 6). The S55V mutant was approximately 2-fold more effective on a molar basis than wild-type hbeta 2m at promoting peptide binding. When binding was examined over a wide range of peptide concentrations, the ED50 values in the presence of an almost saturating concentration of hbeta 2m and S55V (10 µM) were 54- and 77-fold greater, respectively, than observed in the absence of beta 2m (Table I).


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 6.   The S55V mutant augments peptide binding at limiting peptide concentrations relative to wild-type hbeta 2m. Hmy2.C1R-A2 cells were centrifuged and resuspended in serum-free IMDM with 100 ng/ml of the FITC-conjugated HIV RT peptide and titrations of wild-type hbeta 2m (black-diamond ) or S55V (black-square) for 17 h at 37 °C. Samples were washed twice with 2 mg/ml BSA in PBS, resuspended, and analyzed by flow cytometry. All values are expressed as mean fluorescence intensity.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Peptide binding to cell surface HLA-A2: effects of hbeta 2m and S55V
FITC-labeled HIV RT peptide (HLA-A2-restricted) was incubated from 0.457 to 27,000 ng/ml in serum-free medium with Hmy2.C1R-A2 transfectants in the presence or absence of 10 µM hbeta 2m or S55V for 17 h. Samples were then washed twice and analyzed by flow cytometry. ED50 values were calculated using a sigmoid logistic fit.

The augmented ability of the S55V variant to enhance exogenous peptide loading of cells suggested that these cells would also be better targets for CTL recognition. To test this possibility, two CTL clones, specific for an HTLV-1 TAX peptide in the context of HLA-A2 and an influenza nucleoprotein peptide in the context of HLA-A3, were used in a standard lysis assay of Hmy2.C1R transfectants pulsed with a suboptimal concentration of antigenic peptide. The S55V mutant was 4-fold more effective at a molar level than wild-type beta 2m at enhancing target cell lysis for HLA-A2 (Fig. 7a) and 6- to 7-fold better for HLA-A3 (Fig. 7b). Controls with irrelevant HLA-A2 and HLA-A3 binding peptides with the highest concentrations of beta 2m used resulted in only background levels of killing. Additionally, multiple TAX-specific A2-restricted clones displayed similar levels of S55V-enhanced killing relative to wild-type beta 2m (data not shown).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 7.   Enhanced target cell lysis mediated by the S55V mutant. The S55V mutant enhances CTL recognition better than wild-type hbeta 2m. Hmy2.C1R-A2 (a) or Hmy2.C1R-A3 (b) target cells were pulsed with BATD (Europium chelator) for 30 min and washed in SF CTL medium followed by incubation with a suboptimal dose of TAX peptide (9.3 pM) or control A2-binding HIV gag peptide at 1 nM with the indicated concentrations of beta 2m for 90 min (a) or with a suboptimal dose of NP 265-273 peptide (100 pM) or control A3-binding pn2a.A3 peptide at 1 µM with the indicated concentrations of beta 2m for 60 min (b). Target cells were washed and then combined with the TAX-specific HLA-A2-restricted CTL clone N1218 at an effector:target ratio of 4:1 (a) or the NP-specific HLA-A3 restricted CTL clone 2711 at an effector:target ratio of 2:1 (b) for 2 h. 20 µl of supernatant was harvested, added to 200 µl of Europium solution, and read on a Wallac 1234 DELFIA fluorometer. All incubations were done at 37 °C.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

MHC I molecules have evolved to present endogenously derived antigens and are relatively refractory to peptide loading unless beta 2m is present (22, 23, 56-58). Data presented in Figs. 1-3 demonstrate the extent to which beta 2m facilitates peptide loading and MHC stability. In the absence of beta 2m, up to 100-fold higher concentrations of peptide are required to achieve similar levels of peptide loading and target cell lysis. Since peptides can only bind to MHC I molecules already devoid of peptide and since these empty molecules are normally very unstable (23), the mechanism of exogenous beta 2m's effect on peptide binding is probably via the prolongation of the cell surface half-life of empty MHC I molecules. Therefore, if the exogenous beta 2m concentration is high enough or, alternatively, of high enough affinity, then these empty "peptide-receptive" molecules may remain available on the surface long enough for exogenously added peptide to bind. This beta 2m effect may even be more pronounced in vivo when peptide is administered as a vaccine, because diffusion and/or degradation of these peptides would greatly limit the amount available for cell surface binding. Additionally, a higher affinity beta 2m could result in decreased dissociation rates of peptide·MHC complexes (a longer cell surface half-life) and, therefore, an increased antigen density of the relevant antigenic MHC complexes.

The higher affinity of hbeta 2m for murine MHC heavy chains (31) increases the amount and stability of cell surface MHC I complexes that can be exogenously loaded with antigenic peptide relative to mbeta 2m (26). The resulting antigenic peptide·murine MHC·hbeta 2m complexes that are longer lived would be more likely to induce cell-mediated immune responses. In fact, peptide-specific MHC I-restricted CTL have been successfully elicited in mice by in vivo injection of peptide in the presence but not the absence of hbeta 2m (59). Murine beta 2m is far less efficient at stabilizing cell surface MHC I, presumably due to its lower affinity interaction with heavy chains (26, 31). It might therefore be predicted that in vivo immunization of mice with peptide and murine beta 2m would have been much less effective or not effective at all. Additionally, the use of a xenogeneic beta 2m (human) in mice raises the possibility that processing and presentation of hbeta 2m-derived helper epitopes may have contributed to the reported responses. Clearly, contributions of CD4-derived help must be considered in the design of peptide-based vaccines. Currently, in vivo studies are under way to investigate these questions.

Although some studies demonstrate that peptide and beta 2m exchange can be independent or even antagonistic (60-62), these data clearly demonstrate the enhancement of peptide binding by hbeta 2m. It should also be noted that the system reported here employs human beta 2m and human MHC I molecules, while previous reports dealt exclusively with the murine equivalents (62) or hbeta 2m with murine MHC I molecules (60, 61).

There are notable differences between the abilities of hbeta 2m to exchange onto human MHC I molecules versus mbeta 2m exchanging onto murine MHC I molecules. The myc-beta 2m binding experiments in Fig. 4 require 16 h incubations in the presence of peptide to demonstrate significant beta 2m binding. In contrast, comparable binding of mbeta 2m to murine MHC I molecules is observed within 2 h, even in the absence of peptide (26). Another study demonstrated that up to 5% of HLA molecules on normal T cells exchanged directly fluoresceinated hbeta 2m (35). The authors noted that HLA-A2 on transfected C1R lymphoblastoid cells (such as those used in this study) exchanged much less hbeta 2m than did normal human T cells (35). Based on our study, this exchange would likely be greatly increased in the presence of an A2-restricted peptide. The relatively small amount of peptide-independent exchange demonstrated in the human system was in great contrast to that noted for murine cells (35), consistent with our findings (26). Additionally, others have shown that HLA-A3 and HLA-B27 transfected into the TAP-deficient T2 or RMA-S cells are refractory to peptide and beta 2m loading, while the murine alleles H-2Kb and H-2Db are effectively stabilized (63).

If the exogenous loading characteristics of murine and human MHC I molecules are fundamentally different, it may be advantageous or even necessary to use mice transgenic for human MHC I and hbeta 2m (64-70) in the development of human peptide-based vaccine protocols. The synergy of loading peptide in the presence of higher affinity beta 2ms and the requirement of hbeta 2m to induce CTL in mice (59) indicates the importance of testing immunization regimens with native hbeta 2m and higher affinity variants of beta 2m. Therefore, the engineering of higher affinity variants of human beta 2m represents a significant complementary approach to defining immunodominant epitopes and MHC supermotifs for the development of peptide-based vaccines for the treatment of tumors and viruses.

The ability of beta 2m to bind and stabilize MHC I molecules is clearly related to its relative affinity for the MHC I heavy chains (26). For the development of murine vaccination models, the availability of a naturally occurring higher affinity beta 2m (hbeta 2m) provides this essential component (31, 59). In contrast, there are no known naturally occurring beta 2ms available with higher affinity for human MHC I heavy chains than hbeta 2m. The availability of three-dimensional structural data for a number of MHC I molecules as well as our previous studies examining chimeric mbeta 2m/hbeta 2m molecules (26) led to the identification of a specific region and, ultimately, a single residue of beta 2m (serine 55) to mutate in order to increase its affinity for human MHC I heavy chains.

The identification of the S55V mutant was aided in large part by the availability of a three-dimensional crystal structure for MHC I molecules and underscores the importance and utility of advances in the field of structural biology. In the HLA-A2 structure, serine 55 of beta 2m (at the beta 2m/heavy chain interface) is directly adjacent to an ordered water molecule (52).4 Our earlier work suggested that residue changes that promoted protein/protein interactions (e.g. formation of salt bridges) could predictably improve stability between the two MHC I subunits. Since hydrophobic interactions contribute significantly to the overall stability of proteins (71, 72) and the exclusion of water molecules from an otherwise hydrophobic interface would result in a gain of entropy and make the interaction more thermodynamically favorable (72), we mutated the Ser55 residue to the hydrophobic residues valine and isoleucine. By making these variants, we maximized the opportunity to occupy the hydrophobic cavity without sterically hindering the beta 2m/heavy chain interaction.

By four different criteria (MHC complex stabilization, beta 2m binding, peptide binding, and CTL activity), we have demonstrated that the S55V mutant of human beta 2m binds to human MHC I molecules and enhances the loading of antigenic peptide to a greater extent than wild-type hbeta 2m. Moreover, this higher affinity has been shown to be a general feature for the HLA-A alleles, since it has been extended to all three HLA-A alleles studied. The combination of HLA-A1, -A2, and -A3 is represented in 30-70% of the population, depending on factors such as ethnicity and country of origin (73, 74), suggesting that the S55V mutant may find broad clinical utility in combination with antigenic HLA supermotif peptides that have been selected based on their ability to bind multiple HLA alleles (1-4).

It will be interesting to determine the effect the S55V mutant beta 2m will have on the stability of lower affinity peptides. The qualitative and quantitative effects of the higher affinity of S55V may prolong the stability of MHC complexes exogenously loaded with lower affinity antigenic peptides on the cell surface, thereby allowing for the induction of cell-mediated responses to cryptic, subdominant, and marginally antigenic peptides. The generation of these responses could serve to eliminate neoplasms and some virally infected cells (e.g. HIV or herpes) that otherwise might evade detection. Coupled with an appropriate regimen to induce a strong cell-mediated immune response, a patient could be cleared of tumors and acquire protective immunity to viruses without the necessity of immunization with the neoplasms/pathogens themselves. Thus, in concert with efforts identifying antigenic T cell epitopes (5-9) and antigenic MHC supermotif peptides (1-4), hbeta 2m and higher affinity variants may find global utility in the development of effective, novel peptide-based vaccine protocols to treat both tumors and viral infections.

    ACKNOWLEDGEMENTS

We thank Drs. Jonathan Ashwell, Jonathan Yewdell, Charles Zacharchuck, and Allan Weissman for suggestions and critical review of this manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by the NCI, National Institutes of Health (NIH), Biotechnology Training Program.

parallel Supported by the NCI (NIH) Summer Research Training Fellowship Program.

** To whom all correspondence should be addressed: Molecular Applications Group, 11800 Dewey Rd., Silver Spring, MD 20906-4881. Tel.: 301-946-8035; Fax: 301-946-8036, E-mail: rkr{at}mag.com.

The abbreviations used are: MHC, major histocompatibility complex; beta 2m, beta 2-microglobulinhbeta 2m, human beta 2-microglobulinmbeta 2m, murine beta 2-microglobulinCTL, cytotoxic T lymphocyteSF, serum-freeBATD, bis(acetoxymethyl)2,2':6',2"-terpyridine-6',6"-decarboxylateBSA, bovine serum albuminHTLV-1, human T cell lymphotrophic virus-1FITC, fluorescein isothiocyanateAb, antibodymAb, monoclonal antibodyHIV, human immunodeficiency virusIMDM, Iscove's modified Dulbecco's mediumFACS, fluorescence-activated cell sortingPBS, phosphate-buffered salineRT, reverse transcriptase.

2 Available on the World Wide Web at http://cancernet.nci.nih.gov/ prot/menu/dx01302.html.

3 M. J. Shields, unpublished observations.

4 Protein Data Bank (Brookhaven National Laboratory, Upton, NY) code 3HLA.

5 M. J. Shields and R. K. Ribaudo, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. del Guercio, M. F., Sidney, J., Hermanson, G., Perez, C., Grey, H. M., Kubo, R. T., and Sette, A. (1995) J. Immunol. 154, 685-693[Abstract]
  2. Sidney, J., del Guercio, M. F., Southwood, S., Engelhard, V. H., Appella, E., Rammensee, H. G., Falk, K., Rotzschke, O., Takiguchi, M., Kubo, R. T., Grey, H. M., and Sette, A. (1995) J. Immunol. 154, 247-259[Abstract]
  3. Sidney, J., Grey, H. M., Southwood, S., Celis, E., Wentworth, P. A., del Guercio, M. F., Kubo, R. T., Chesnut, R. W., and Sette, A. (1996) Hum. Immunol. 45, 79-93[CrossRef][Medline] [Order article via Infotrieve]
  4. Sidney, J., Southwood, S., del Guercio, M. F., Grey, H. M., Chesnut, R. W., Kubo, R. T., and Sette, A. (1996) J Immunol 157, 3480-3490[Abstract]
  5. Zhang, C., Cornette, J. L., Berzofsky, J. A., and DeLisi, C. (1997) Vaccine 15, 1291-1302[CrossRef][Medline] [Order article via Infotrieve]
  6. Deng, Y., Yewdell, J. W., Eisenlohr, L. C., and Bennink, J. R. (1997) J. Immunol. 158, 1507-1515[Abstract]
  7. Eberl, G., Kessler, B., Eberl, L. P., Brunda, M. J., Valmori, D., and Corradin, G. (1996) Eur. J. Immunol. 26, 2709-2716[Medline] [Order article via Infotrieve]
  8. Evans, D. T., Piekarczyk, M. S., Allen, T. M., Boyson, J. E., Yeager, M., Hughes, A. L., Gotch, F. M., Hinshaw, V. S., and Watkins, D. I. (1997) J. Immunol. 159, 1374-1382[Abstract]
  9. Man, S., Ridge, J. P., and Engelhard, V. H. (1994) J. Immunol. 153, 4458-4467[Abstract]
  10. Germain, R. N., and Margulies, D. H. (1993) in Annual Review of Immunology (Paul, W. P., Fathman, C. G., and Metzger, H., eds), Vol. 11, pp. 402-450, Annual Reviews Inc., Palo Alto, CA
  11. Yewdell, J. W., and Bennink, J. R. (1992) Adv. Immunol. 52, 1-123[Medline] [Order article via Infotrieve]
  12. Hansen, T. H., and Lee, D. R. (1997) Adv. Immunol. 64, 105-137[Medline] [Order article via Infotrieve]
  13. Montgomery, D. L., Ulmer, J. B., Donnelly, J. J., and Liu, M. A. (1997) Pharmacol. Ther. 74, 195-205[CrossRef][Medline] [Order article via Infotrieve]
  14. Manickan, E., Karem, K. L., and Rouse, B. T. (1997) Crit. Rev. Immunol. 17, 139-154[Medline] [Order article via Infotrieve]
  15. Shiver, J. W., Davies, M. E., Yasutomi, Y., Perry, H. C., Freed, D. C., Letvin, N. L., and Liu, M. A. (1997) Vaccine 15, 884-887[CrossRef][Medline] [Order article via Infotrieve]
  16. Suhrbier, A. (1997) Immunol. Cell Biol. 75, 402-408[Medline] [Order article via Infotrieve]
  17. Reis e Sousa, C., and Germain, R. N. (1995) J. Exp. Med. 182, 841-851[Abstract/Free Full Text]
  18. Day, P. M., Yewdell, J. W., Porgador, A., Germain, R. N., and Bennink, J. R. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8064-8069[Abstract/Free Full Text]
  19. Weiskirch, L. M., and Paterson, Y. (1997) Immunol. Rev. 158, 159-169[CrossRef][Medline] [Order article via Infotrieve]
  20. Moss, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11341-11348[Abstract/Free Full Text]
  21. Zhu, X., Tommasino, M., Vousden, K., Sadovnikava, E., Rappuoli, R., Crawford, L., Kast, M., Melief, C. J., Beverley, P. C., and Stauss, H. J. (1995) Scand. J. Immunol. 42, 557-563[CrossRef][Medline] [Order article via Infotrieve]
  22. Vitiello, A., Potter, T. A., and Sherman, L. A. (1990) Science 250, 1423-1426[Abstract/Free Full Text]
  23. Kozlowski, S., Takeshita, T., Boehncke, W. H., Takahashi, H., Boyd, L. F., Germain, R. N., Berzofsky, J. A., and Margulies, D. H. (1991) Nature 349, 74-77[CrossRef][Medline] [Order article via Infotrieve]
  24. Ljunggren, H. G., Stam, N. J., Ohlen, C., Neefjes, J. J., Hoglund, P., Heemels, M. T., Bastin, J., Schumacher, T. N., Townsend, A., Karre, K., and Plough, H. (1990) Nature 346, 476-480[CrossRef][Medline] [Order article via Infotrieve]
  25. Otten, G. R., Bikoff, E., Ribaudo, R. K., Kozlowski, S., Margulies, D. H., and Germain, R. N. (1992) J. Immunol. 148, 3723-3732[Abstract]
  26. Shields, M. J., Assefi, N. P., Hodgson, W., Kim, E. J., and Ribaudo, R. K. (1998) J. Immunol. 160, 2297-2307[Abstract/Free Full Text]
  27. Abastado, J. P., Casrouge, A., and Kourilsky, P. (1993) J. Immunol. 151, 3569-3575[Abstract]
  28. Jaulin, C., Romero, P., Luescher, I. F., Casanova, J. L., Prochnicka-Chalufour, A., Langlade-Demoyen, P., Maryanski, J. L., and Kourilsky, P. (1992) Int. Immunol. 4, 945-953[Abstract/Free Full Text]
  29. Ribaudo, R. K., and Margulies, D. H. (1995) J. Immunol. 155, 3481-3493[Abstract]
  30. Fukazawa, T., Hermann, E., Edidin, M., Wen, J., Huang, F., Kellner, H., Floege, J., Farahmandian, D., Williams, K. M., and Yu, D. T. (1994) J. Immunol. 153, 3543-3550[Abstract]
  31. Pedersen, L. O., Stryhn, A., Holter, T. L., Etzerodt, M., Gerwien, J., Nissen, M. H., Thogersen, H. C., and Buus, S. (1995) Eur. J. Immunol. 25, 1609-1616[Medline] [Order article via Infotrieve]
  32. Tatake, R. J., and Zeff, R. A. (1993) Immunogenetics 38, 318-322[Medline] [Order article via Infotrieve]
  33. Trymbulak, W. P., and Zeff, R. A. (1997) Immunogenetics 46, 418-426[CrossRef][Medline] [Order article via Infotrieve]
  34. Trymbulak, W. P. J., and Zeff, R. A. (1997) Transplantation 64, 640-645[CrossRef][Medline] [Order article via Infotrieve]
  35. Edidin, M., Achilles, S., Zeff, R., and Wei, T. (1997) Immunogenetics 46, 41-45[CrossRef][Medline] [Order article via Infotrieve]
  36. Bremers, A. J., van der Burg, S. H., Kuppen, P. J., Kast, W. M., van de Velde, C. J., and Melief, C. J. (1995) J. Immunother. Emphasis Tumor Immunol. 18, 77-85[Medline] [Order article via Infotrieve]
  37. van der Burg, S. H., Ras, E., Drijfhout, J. W., Benckhuijsen, W. E., Bremers, A. J., Melief, C. J., and Kast, W. M. (1995) Hum. Immunol. 44, 189-198[CrossRef][Medline] [Order article via Infotrieve]
  38. Shields, M. J., and Ribaudo, R. K. (1998) Tissue Antigens 51, 567-570[Medline] [Order article via Infotrieve]
  39. Storkus, W. J., Howell, D. N., Salter, R. D., Dawson, J. R., and Cresswell, P. (1987) J. Immunol. 138, 1657-1659[Medline] [Order article via Infotrieve]
  40. Winter, C. C., Carreno, B. M., Turner, R. V., Koenig, S., and Biddison, W. E. (1991) J. Immunol. 146, 3508-3512[Abstract]
  41. DiBrino, M., Tsuchida, T., Turner, R. V., Parker, K. C., Coligan, J. E., and Biddison, W. E. (1993) J. Immunol. 151, 5930-5935[Abstract]
  42. Biddison, W. E., Kubota, R., Kawanishi, T., Taub, D. D., Cruikshank, W. W., Center, D. M., Connor, E. W., Utz, U., and Jacobson, S. (1997) J. Immunol. 159, 2018-2025[Abstract]
  43. Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985) Mol. Cell. Biol. 5, 3610-3616[Abstract/Free Full Text]
  44. DiBrino, M., Parker, K. C., Shiloach, J., Knierman, M., Lukszo, J., Turner, R. V., Biddison, W. E., and Coligan, J. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1508-1512[Abstract/Free Full Text]
  45. DiBrino, M., Parker, K. C., Shiloach, J., Turner, R. V., Tsuchida, T., Garfield, M., Biddison, W. E., and Coligan, J. E. (1994) J. Immunol. 152, 620-631[Abstract]
  46. Honma, K., Parker, K. C., Becker, K. G., McFarland, H. F., Coligan, J. E., and Biddison, W. E. (1997) J. Neuroimmunol. 73, 7-14[CrossRef][Medline] [Order article via Infotrieve]
  47. Parker, K. C., Bednarek, M. A., Hull, L. K., Utz, U., Cunningham, B., Zweerink, H. J., Biddison, W. E., and Coligan, J. E. (1992) J. Immunol. 149, 3580-3587[Abstract]
  48. Parker, K. C., Bednarek, M. A., and Coligan, J. E. (1994) J. Immunol. 152, 163-175[Abstract]
  49. Parker, K. C., Shields, M., DiBrino, M., Brooks, A., and Coligan, J. E. (1995) Immunol. Res. 14, 34-57[Medline] [Order article via Infotrieve]
  50. Depierreux, C., Graff, I., Lancelot, V., Denis, M., Vanderplasschen, A., Hanon, E., Pastoret, P. P., and Swysen, C. (1997) J. Immunol. Methods 203, 77-88[CrossRef][Medline] [Order article via Infotrieve]
  51. Sugawara, S., Abo, T., and Kumagai, K. (1987) J. Immunol. Methods 100, 83-90[CrossRef][Medline] [Order article via Infotrieve]
  52. Bjorkman, P. J., Saper, M. A., Samraoui, B., Bennett, W. S., Strominger, J. L., and Wiley, D. C. (1987) Nature 329, 506-512[CrossRef][Medline] [Order article via Infotrieve]
  53. Brodsky, F. M., and Parham, P. (1982) J. Immunol. 128, 129-135[Abstract]
  54. Ribaudo, R. K., and Margulies, D. H. (1992) J. Immunol. 149, 2935-2944[Abstract]
  55. Hochman, J. H., Shimizu, Y., DeMars, R., and Edidin, M. (1988) J. Immunol. 140, 2322-2329[Abstract]
  56. Rock, K. L., Rothstein, L. E., Gamble, S. R., and Benacerraf, B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7517-7521[Abstract/Free Full Text]
  57. Kane, K. P., Sherman, L. A., and Mescher, M. F. (1991) Eur. J. Immunol. 21, 2289-2292[Medline] [Order article via Infotrieve]
  58. Rock, K. L., Gamble, S., Rothstein, L., and Benacerraf, B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 301-304[Abstract/Free Full Text]
  59. Rock, K. L., Fleischacker, C., and Gamble, S. (1993) J. Immunol. 150, 1244-1252[Abstract]
  60. Smith, J. D., Lie, W. R., Gorka, J., Myers, N. B., and Hansen, T. H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7767-7771[Abstract/Free Full Text]
  61. Cook, J. R., Myers, N. B., and Hansen, T. H. (1996) J. Immunol. 157, 2256-2261[Abstract]
  62. Horig, H., Papadopoulos, N. J., Vegh, Z., Palmieri, E., Angeletti, R. H., and Nathenson, S. G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13826-13831[Abstract/Free Full Text]
  63. Anderson, K. S., Alexander, J., Wei, M., and Cresswell, P. (1993) J. Immunol. 151, 3407-3419[Abstract]
  64. Barra, C., Gournier, H., Garcia, Z., Marche, P. N., Jouvin-Marche, E., Briand, P., Fillipi, P., and Lemonnier, F. A. (1993) J. Immunol. 150, 3681-3689[Abstract]
  65. Danliczyk, U. G., and Delovitch, T. L. (1994) J. Immunol. 153, 3533-3542[Abstract]
  66. Epstein, H., Hardy, R., May, J. S., Johnson, M. H., and Holmes, N. (1989) Eur. J. Immunol. 19, 1575-1583[Medline] [Order article via Infotrieve]
  67. Khare, S. D., Luthra, H. S., and David, C. S. (1995) J. Exp. Med. 182, 1153-1158[Abstract/Free Full Text]
  68. Lustgarten, J., Theobald, M., Labadie, C., LaFace, D., Peterson, P., Disis, M. L., Cheever, M. A., and Sherman, L. A. (1997) Hum. Immunol. 52, 109-118[CrossRef][Medline] [Order article via Infotrieve]
  69. Taurog, J. D., Maika, S. D., Simmons, W. A., Breban, M., and Hammer, R. E. (1993) J. Immunol. 150, 4168-4178[Abstract]
  70. Vitiello, A., Marchesini, D., Furze, J., Sherman, L. A., and Chesnut, R. W. (1991) J. Exp. Med. 173, 1007-1015[Abstract/Free Full Text]
  71. Waldburger, C. D., Jonsson, T., and Sauer, R. T. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2629-2634[Abstract/Free Full Text]
  72. Branden, C., and Tooze, J. (eds) (1991) Introduction to Protein Structure, pp. 247-268, Garland Publishing, Inc., New York
  73. Schipper, R. F., D'Amaro, J., Bakker, J. T., Bakker, J., van Rood, J. J., and Oudshoorn, M. (1997) Hum. Immunol. 52, 54-71[CrossRef][Medline] [Order article via Infotrieve]
  74. Mori, M., Beatty, P. G., Graves, M., Boucher, K. M., and Milford, E. L. (1997) Transplantation 64, 1017-1027[Medline] [Order article via Infotrieve]

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Ann Rheum DisHome page
J-E Gottenberg, M Busson, J Cohen-Solal, F Lavie, K Abbed, R P Kimberly, J Sibilia, and X Mariette
Correlation of serum B lymphocyte stimulator and {beta}2 microglobulin with autoantibody secretion and systemic involvement in primary Sjogren's syndrome
Ann Rheum Dis, July 1, 2005; 64(7): 1050 - 1055.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. Berko, Y. Carmi, G. Cafri, S. Ben-Zaken, H. M. Sheikhet, E. Tzehoval, L. Eisenbach, A. Margalit, and G. Gross
Membrane-Anchored {beta}2-Microglobulin Stabilizes a Highly Receptive State of MHC Class I Molecules
J. Immunol., February 15, 2005; 174(4): 2116 - 2123.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. Laugel, J. M. Boulter, N. Lissin, A. Vuidepot, Y. Li, E. Gostick, L. E. Crotty, D. C. Douek, J. Hemelaar, D. A. Price, et al.
Design of Soluble Recombinant T Cell Receptors for Antigen Targeting and T Cell Inhibition
J. Biol. Chem., January 21, 2005; 280(3): 1882 - 1892.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Gidalevitz, C. Biswas, H. Ding, D. Schneidman-Duhovny, H. J. Wolfson, F. Stevens, S. Radford, and Y. Argon
Identification of the N-terminal Peptide Binding Site of Glucose-regulated Protein 94
J. Biol. Chem., April 16, 2004; 279(16): 16543 - 16552.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. M. Hill, T. Kasliwal, E. Schwarz, A. M. Hebert, T. Chen, E. Gubina, L. Zhang, and S. Kozlowski
A Dominant Negative Mutant beta 2-Microglobulin Blocks the Extracellular Folding of a Major Histocompatibility Complex Class I Heavy Chain
J. Biol. Chem., February 14, 2003; 278(8): 5630 - 5638.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Glick, D. A. Price, A.-L. Vuidepot, T. B. Andersen, S. L. Hutchinson, B. Laugel, A. K. Sewell, J. M. Boulter, P. R. Dunbar, V. Cerundolo, et al.
Novel CD8+ T Cell Antagonists Based on beta 2-Microglobulin
J. Biol. Chem., May 31, 2002; 277(23): 20840 - 20846.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. Michaelsson, A. Achour, A. Rolle, and K. Karre
MHC Class I Recognition by NK Receptors in the Ly49 Family Is Strongly Influenced by the {{beta}}2-Microglobulin Subunit
J. Immunol., June 15, 2001; 166(12): 7327 - 7334.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shields, M. J.
Right arrow Articles by Ribaudo, R. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shields, M. J.
Right arrow Articles by Ribaudo, R. K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 1998 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement