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Disulfide Bond Engineering to Trap Peptides in the MHC Class I Binding Groove¹

Steven M. Truscott^*, Lonnie Lybarger, John M. Martinko, Vesselin E. Mitaksov^*, David M. Kranz, Janet M. Connolly^*, Daved H. Fremont^* and Ted H. Hansen^2,*

^* Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110; Cell Biology and Anatomy, University of Arizona Health Sciences Center, Tucson, AZ 85724; Microbiology, Southern Illinois University, Carbondale, IL 62901; and Biochemistry, University of Illinois, Urbana, IL 61801

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Immunodominant peptides in CD8 T cell responses to pathogens and tumors are not always tight binders to MHC class I molecules. Furthermore, antigenic peptides that bind weakly to the MHC can be problematic when designing vaccines to elicit CD8 T cells in vivo or for the production of MHC multimers for enumerating pathogen-specific T cells in vitro. Thus, to enhance peptide binding to MHC class I, we have engineered a disulfide bond to trap antigenic peptides into the binding groove of murine MHC class I molecules expressed as single-chain trimers or SCTs. These SCTs with disulfide traps, termed dtSCTs, oxidized properly in the endoplasmic reticulum, transited to the cell surface, and were recognized by T cells. Introducing a disulfide trap created remarkably tenacious MHC/peptide complexes because the peptide moiety of the dtSCT was not displaced by high-affinity competitor peptides, even when relatively weak binding peptides were incorporated into the dtSCT. This technology promises to be useful for DNA vaccination to elicit CD8 T cells, in vivo study of CD8 T cell development, and construction of multivalent MHC/peptide reagents for the enumeration and tracking of T cells—particularly when the antigenic peptide has relatively weak affinity for the MHC.

	Introduction

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A critical threshold density of cell surface peptide/MHC complexes (pMHC)³ is required to prime naive CD8 T cells and to activate cytolytic CD8 effectors. Although the affinity of the peptide for the MHC is a major factor in determining the immunogenicity of any particular peptide Ag, immunodominant peptides in CD8 T cell responses to pathogens are not always the tightest MHC binders (1). This is because immunodominance is also influenced by levels of donor protein in the cytosol, by the relative efficiency of processing particular epitopes, and by pathogen interference with Ag presentation. Moreover, the T cell repertoire has a major impact on determining which peptides are immunodominant in CD8 T cell responses (1, 2, 3). The fact that certain antigenic peptides are difficult to process or are relatively weak binders has considerable implications for vaccination strategies against pathogens and tumors (4). Poor peptide binding is particularly problematic for primary CD8 T cell responses that require cross-priming. In the case of cross-priming, professional APCs that have acquired Ag exogenously, rather than by continuing endogenous synthesis in the cytosol, are only capable of activating T cells if pMHC complexes have a sufficient half-life (5).

Tetramers and other multivalent staining reagents used to enumerate CD8 T cell responses to pathogens and tumors are also highly influenced by class I peptide-binding affinity. Such reagents are typically made with soluble recombinant class I H chains expressed in bacteria and refolded with synthetic peptides (6). The production and stability of tetramers is critically determined by the affinity of the peptide for the MHC, thus limiting the study of T cells specific for lower affinity complexes. A technology for stabilizing these recombinant pMHC complexes would allow for more rigorous characterizations of CD8 T cell responses elicited by the lower affinity determinants. Although a number of these are immunodominant, they are more often subdominant. However, the importance of subdominant determinants cannot be understated, as they have been found to be protective in vaccine strategies (7, 8, 9).

To bypass the inefficiencies of Ag processing and to potentially enhance the binding of weak antigenic peptides, we engineered MHC class I molecules as single-chain trimers (SCTs) (10). SCTs have the format: secretion signal sequence–antigenic peptide–[G₄S]₃–mature ₂-microglobulin (₂m)–[G₄S]₄–mature class I H chain ([G₄S]_n, n multiples of Gly-Gly-Gly-Gly-Ser) (see Fig. 1A). To characterize peptide binding by the SCT, we have focused most of our attention on the SCT construct of the well-characterized H-2K^b/OVA_257–264 (K^b/OVA) complex. This SCT was found to have remarkable qualities, including extended cell surface half-life and the ability to potently stimulate T cells that recognize native K^b/OVA (10). Furthermore, this SCT was found to be 1000-fold more refractory to exogenous peptide binding compared with K^b loaded with endogenous peptides. However, compared with K^b preloaded with OVA peptide, the SCT was more susceptible to exogenous peptide binding (11). This latter finding demonstrated that binding of the SCT peptide was likely impaired by the first Gly-Ser linker of the SCT, which extends beyond the peptide binding groove, thus disrupting interactions in the H chain F pocket, which normally anchors the C terminus of the peptide. Since OVA is a particularly high-affinity peptide ligand for K^b (12), it is able to compensate for disruption of F pocket anchoring. This explains the improved cell surface stability of the OVA.₂m.K^b SCT vs native K^b/OVA: the SCT is able to quickly rebind the covalently attached high-affinity OVA peptide. Consistent with this finding, we also determined that SCTs constructed with tight binding peptides were able to attain higher levels of steady-state folding and surface expression compared with SCTs constructed with weak binding peptides (13). Therefore, to re-engineer the SCT so that the peptide remained bound and to enhance the binding of weaker peptides, we investigated the possibility of strategically positioning a disulfide bond in the SCT.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. A structure-based approach to engineering a disulfide bond in single-chain MHC class I. A, Schematic representation of the SCT based on the published structure for Kb/OVA (PDB code: 1VAC), including Gly-Ser linkers (red) and hypothetical location of the engineered disulfide bond (green). B, Analysis of superimposed class I and class II structures to select residues for cysteine mutagenesis. Kb H chain (blue) aligned with the I-Ek-bound hemoglobin peptide (yellow, PDB code: 1FNE), which features covalent attachment with a Gly-Ser linker to the -chain of class II. This structure-based visual provided a framework for examining distances between residues on the class I H chain and along the first SCT linker. Three residues on the Kb H chain were selected for mutagenesis (purple): T80, Y84, and N86. Appropriately spaced residues in the linker were matched with each of these three H chain positions (see Table I and Fig. 2). The second linker position was chosen for further characterization of dtSCT constructs (Y84C, L2C).

	Materials and Methods

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Cell lines and Abs

Triple-knockout fibroblasts (K^b–/–D^b–/–₂m^–/–; 3KO) are a transformed murine embryo fibroblast line derived from 3KO mice (20). B6/WT3 (21) is a C57BL/6 (H-2^b) murine embryo fibroblast line and was a gift from S. Jennings (Louisiana State University Health Sciences Center, Shreveport, LA). LM1.8 cells are L cells (H-2^k) transfected with ICAM (22) and were a gift from Dr. P. Kourilsky (Institut National de la Santé et de la Recherche Médicale, Institut Pasteur, Paris, France). mAbs used in this study include the following: B8-24-3, which recognizes folded K^b (American Type Culture Collection); Y3, which recognizes folded H-2K molecules (23); 25-D1.16 (a gift from Dr. J. Yewdell, National Institutes of Health, Bethesda, MD), which recognizes K^b plus SIINFEKL peptide (24); and 64-3-7, which recognizes open (peptide-free) forms of L^d and other class I molecules tagged with the 64-3-7 epitope (25).

Peptides

The OVA_257–264 peptide (SIINFEKL) (26), SIYR peptide (SIYRYYGL) (27), VSV8 peptide (RGYVYQGL) (28), the QL9 peptide (QLSPFPFDL) (29, 30, 31), and the murine CMV (MCMV) pp89 peptide (YPHFMPTNL) (32) were synthesized on an Applied Biosystems Model 432A peptide synthesizer. Peptides were dissolved directly in culture medium and incubated with cells for 5 h or overnight before cytofluorometry.

DNA constructs and retroviral transduction

Constructs were generated using standard techniques and confirmed by DNA sequence analysis. The OVA.₂m.K^b SCT sequence was previously described (10), and the QL9.₂m.L^d SCT was constructed similarly as follows. The secretion signal sequence of ₂m^b is followed immediately by the peptide sequence, then a linker of 15 residues, [G₄S]₃. An alanine residue necessarily occupies the fourth position of the first linker (Table I) as a result of the placement of a convenient restriction site in the SCT construct. This first linker is followed by the mature ₂m^b sequence, the second linker of 20 residues, [G₄S]₄, then the mature class I sequence. The Y84A mutation, first described in the OVA.₂m.K^b SCT (11), was also introduced into the QL9.₂m.L^d SCT. This mutation and the cysteine mutations shown in Table I were introduced by site-directed mutagenesis (QuikChange II XL; Stratagene). Retroviral expression vectors pMSCV-IRES-hygromycin and pMSCV-IRES-neomycin were constructed in our lab (33) and used for concomitant expression of class I SCTs and drug resistance genes. Retrovirus-containing supernatants were generated using the Vpack vector system (Stratagene) for transient transfection of 293T cells to generate ecotropic virus for infection of 3KO and B6/WT3 cells or amphitropic virus for infection of LM1.8 cells.

Viable cells, gated by forward and side scatter, were analyzed on a FACSCalibur (BD Biosciences), and data (10,000 events/sample) were analyzed using CellQuest software (BD Biosciences). Staining with anti-class I mAbs was visualized using PE-conjugated goat anti-mouse IgG (BD Biosciences). Soluble, recombinant m6 TCR was used for flow cytometry directly from the supernatant of insect cells (34, 35, 36). Secondary reagents used to detect binding of the m6 TCR included biotinylated anti-mouse TCR -chain mAb H57 (BD Biosciences) and PE-conjugated streptavidin (BD Biosciences).

Immunoprecipitation and Western blotting

Immunoprecipitations were performed essentially as described previously (20). Briefly, cells were treated with 1.0% Nonidet P-40 (IGEPAL CA-630; Sigma-Aldrich) lysis buffer, including 20 mM iodoacetamide, protease inhibitors, and a saturating concentration of precipitating mAb. After 30 min on ice, postnuclear lysates were incubated with protein A-Sepharose beads and washed four times with 0.1% Nonidet P-40 in PBS. Lithium dodecyl sulfate sample buffer (Invitrogen Life Technologies) was added to the beads, and 2-ME (Sigma-Aldrich) was either added to a final concentration of 1% or excluded for nonreducing gels. Western blotting was performed after SDS-PAGE separation of precipitated proteins as described previously (37). Rabbit serum for blotting K^b was generated in our lab by peptide immunization using an amino acid sequence from the cytoplasmic tail of K^b. Rabbit serum for detecting ₂m was also generated in our lab by immunizing with denatured, recombinant mouse ₂m. Biotin-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories) was used as a secondary staining reagent, followed by streptavidin-HRP (Zymed Laboratories). Biotinylated mAb 64-3-7 was used for blotting L^d. Specific proteins were visualized by chemiluminescence with the ECL System (Amersham Biosciences).

CTL assays

LM1.8 target cells transduced with the indicated class I constructs were labeled with Na⁵¹CrO₄ for 1 h. OT-1 or 2C T cells were plated at various concentrations onto 96-well microtiter plates and incubated with target cells for 4 h at 37°C in 5% CO₂ with or without 10 µM exogenous peptide. Radioactivity in supernatants was measured in an Isomedic gamma counter (ICN Biomedicals). The mean of triplicate samples was calculated, and percentage ⁵¹Cr release was determined as follows: percentage of ⁵¹Cr release = 100 x ((experimental ⁵¹Cr release – control ⁵¹Cr release)/(maximum ⁵¹Cr release – control ⁵¹Cr release)), where experimental ⁵¹Cr release represents counts from target cells mixed with effector cells, control ⁵¹Cr release represents counts from target cells in medium alone, and maximum ⁵¹Cr release represents counts from target cells lysed with 5% (v/v) Triton X-100 (Sigma-Aldrich).

N15 hybridoma assays

The TCR^– murine T cell hybridoma 58^–– (38) transfected with the - and -chains for the N15 TCR was a gift from Dr. H.-C. Chang (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA). This cell line is also transfected with CD3 and CD8 cDNAs to optimize TCR expression and recognition of class I (39). Biological activity of IL-2 secreted by the N15 hybridoma was used to detect K^b/VSV8 complexes. B6/WT3 cells or 3KO cells (5 x 10⁴/200 µl/well) expressing endogenous K^b or K^b SCT constructs were incubated for 1 h at 37°C with the indicated concentrations of VSV8 peptide in a flat-bottom 96-well plate. The cells were washed, fixed in 1% paraformaldehyde for 15 min at room temperature, then washed again. The N15 hybridoma cells (10⁵/200 µl/well) in fresh medium were then added to the plate and cultured for 24 h. Supernatants were harvested and frozen at –80°C for at least 1 h to lyse trace cells that may have carried over. CTLL-2 cells were washed and added to supernatants at a final cell density of 10⁴/200 µl/well in a 96-well plate. After 18–24 h of incubation, Alamar blue (BioSource International) was added at 20 µl/well, and relative amounts of IL-2 in each supernatant were determined by fluorescence on a multidetection microplate reader (Bio-Tek Instruments).

	Results

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Disulfide bond engineering strategy

To secure the SCT peptide firmly in the class I peptide-binding groove, we set out to covalently link the peptide directly to the H chain 1 helix using a disulfide bond. As in our previous characterization of SCT molecules (10, 11), we again chose the K^b/OVA system, for which there are excellent reagents to monitor both MHC conformation and peptide occupancy (24, 40). Our approach was to introduce cysteine residues by site-directed mutagenesis, one in the H chain of K^b and one in the first linker (Fig. 1A), which extends from the C terminus of the SIINFEKL peptide to the N terminus of mature ₂m. Placing the second cysteine in the linker, rather than within the peptide itself, we hypothesized would minimize perturbations to the pMHC conformation and also allow us to more readily translate this disulfide-bonding approach to SCT constructs based on other pMHC complexes.

To select the exact positions for cysteine mutations, we superimposed the crystal structures of K^b/OVA (41) and the MHC class II molecule I-E^k bound to a hemoglobin peptide (Hb_64–76) (42). This latter structure features the Hb_64–76 peptide covalently attached with a Gly-Ser linker to the -chain of class II (43, 44). The linker extends from the open class II groove from the C-terminal residue of the Hb_64–76 peptide. Thus, overlaying these structures provided a framework for determining distances between K^b H chain residues and specific positions along the first SCT linker (Fig. 1B). Three residues on the K^b H chain were selected for substitution of a cysteine residue: Thr⁸⁰, Tyr⁸⁴, and Asn⁸⁶. Importantly, the Y84A H chain mutation was also incorporated into the SCTs with the T80C and N86C mutations since we previously found that the Y84A mutation allowed for better linker accommodation (11). Based on the superimposition shown in Fig. 1B, the locations for various linker cysteine residues were selected. Thus, several constructs were made and tested for potential disulfide bond formation to trap the peptide into the class I-binding groove (Table I). SCT mutants with single cysteine mutations at the three abovementioned H chain positions were also constructed to control for the consequences of having an unpaired cysteine.

Characterization of SCT cysteine variants

Retroviral transduction was used to introduce each of the SCT variants into 3KO (20). This permitted the unambiguous analysis of the transduced class I constructs. It should be noted that cell surface expression of SCTs with disulfide traps was also confirmed in wild-type cells (see Fig. 5). Each of the SCT cysteine variants was detected at the cell surface by flow cytometry (Fig. 2A), both with mAb B8-24-3, which detects folded, peptide-occupied K^b, and with mAb 25-D1.16, which binds specifically to the K^b/OVA complex (24). K^b-reactive mAb Y3 also recognized the SCT variants (data not shown). The engineered molecules were expressed at high levels similar to the control SCT Y84A, suggesting that they had folded properly in the ER, passed ER quality control mechanisms, and efficiently transited to the cell surface. Importantly, the level of staining with the K^b/OVA-specific Ab was constant relative to the amount of folded K^b with the exception of the control SCT variant T80C. The mAb 25-D1.16 is known to bind near the C terminus of the peptide (45), and thus, the unpaired cysteine or the loss of threonine at position 80 could impair mAb 25-D1.16 detection of this variant SCT. However, when the T80C H chain mutation was combined with a cysteine mutation in the linker, efficient recognition by mAb 25-D1.16 was restored. The comparable detection of the other variant SCTs with mAbs 25-D1.16 and B8-24-3 suggests that the introduction of cysteines at these various locations in the SCT spacer and/or H chain had no significant impact on the pMHC conformation detected by mAb 25-D1.16.

View larger version (36K): [in this window] [in a new window]   FIGURE 5. Serological analysis of native Ld and QL9.2m.Ld SCT and dtSCT constructs. B6/WT3 (H-2b) fibroblasts (A) were transduced with native Ld (B), QL9.2m.Ld (first-generation SCTs) (C), QL9.2m.Ld Y84A (second-generation SCTs) (D), or QL9.2m.Ld dtSCT (third-generation SCTs) (E). These cells were stained and analyzed by flow cytometry with a panel of three different reagents: mAb 30-5-7 (thin black line), specific for folded (peptide-occupied) Ld; mAb 64-3-7 (shaded in light gray), specific for open (peptide-empty) Ld; and the m6 TCR (thick dark gray line), a soluble high-affinity rTCR reagent specific for the Ld/QL9 complex. Background staining with secondary Ab alone is shown (dotted black line). Along with the label for each histogram is the ratio of open class I conformers to folded conformers (mAb 64-3-7 MFI/mAb 30-5-7 MFI). Equal numbers of cells for each cell line were subjected to immunoprecipitation (IP) and blotting to determine how much of the SCT proteins remained sensitive to EndoH (F), as well as how much endogenous 2m associated with the SCT proteins, relative to the native Ld molecule (G).

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Cell surface expression and gel migration of OVA.2m.Kb SCTs with and without cysteine mutations. A, Fibroblasts derived from mice deficient in 2m, Kb, and Db (3KO cells) were transduced with constructs mutated at specific positions along the class I H chain (columns) and/or along the first Gly-Ser linker of the SCT (rows) (see Table I). The cells were stained with Abs to Kb (mAb B8-24-3, thin black line), the Kb/OVA complex (mAb 25-D1.16, thick gray line), or secondary Ab alone (dotted line) and analyzed by flow cytometry. B, 3KO cells expressing the indicated cysteine mutants were lysed and immunoprecipitated with an Ab to Kb (mAb B8-24-3). These samples were reduced or not reduced and subjected to SDS-PAGE. Western blotting with rabbit serum specific for Kb revealed the migration pattern of the SCTs. Faster migration in the nonreducing gel indicated the formation of an additional disulfide bond.

For a number of reasons, we focused our subsequent analyses on the Y84C, L2C disulfide bond position. First, we had previously engineered position 84 in the SCT H chain, and T cells recognized the Y84A mutant of the SCT, perhaps even more efficiently (11). Second, position 84 is highly conserved among class I alleles, increasing the likelihood that engineering at that position would easily transfer to other class I molecules (see Figs. 5 and 6). Furthermore, it was anticipated that placement of a disulfide bond at position 84 would substitute for hydrogen bonding between the C terminus of the peptide and Y84 in the F pocket of native class I molecules. We chose the second linker position (L2C) downstream of the SCT peptide to match with Y84C, reasoning that a disulfide bond closer to the SCT peptide would provide the best anchor. Thus, the Y84C, L2C construct was chosen for further study and designated dtSCT.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. A, Induction of Ld and Ld SCT cell surface expression by addition of exogenous peptide. As a measure of peptide receptiveness, surface induction was measured for each of the four indicated constructs expressed in B6/WT3 cells. In contrast to previous assays (see Fig. 4), which measured peptide exchange at the cell surface, brefeldin A was excluded from this assay to allow arrival of newly synthesized molecules at the cell surface. Cells were incubated for 4 h with increasing concentrations of MCMV peptide and stained with Ab to Ld (mAb 30-5-7). MFI is plotted relative to the lowest MCMV concentration for each cell line. B, Displacement of the QL9 peptide moiety of QL9.2m.Ld SCT or dtSCT with high-affinity exogenous competitor peptide. B6/WT3 cells expressing the indicated constructs were incubated with increasing concentrations of exogenous MCMV peptide and analyzed by flow cytometry with the high-affinity, recombinant m6 TCR (specific for the Ld/QL9 complexes). MFI at each dose of competitor is plotted as a percentage of the starting signal (no competitor).

Given that T cells are highly sensitive to perturbations in pMHC structure and/or conformation, it was imperative to determine whether the mutations in the dtSCT constructs affected T cell recognition. The dtSCT and each of the previous versions of the OVA.₂m.K^b SCT were transduced into LM1.8 cells (H-2^k fibroblast expressing ICAM) and tested in chromium release assays as targets for K^b/OVA-reactive OT-1 T cells (Fig. 3A). Cells expressing dtSCT and SCT constructs were strongly recognized by OT-1 in cytolytic assays while LM1.8-K^b cells were not. Additionally, the B3Z T cell hybridoma (49), which is also specific for the K^b/OVA complex, responded robustly to 3KO fibroblasts expressing the dtSCT construct (Y84C, L2C) or any of the other cysteine variants (data not shown). Thus, disulfide bond engineering of the single-chain class I molecule preserved the ability to present the OVA epitope to two of two distinct T cell clones. As further evidence of the integrity of the interaction between TCR and SCT molecules, we have found that SCT tetramers and conventional tetramers detect the same polyclonal Ag-specific T cells elicited by pathogen infection; moreover, the same holds true for tetramers built with the engineered disulfide trap—they retain the ability to bind the same T cells as conventional tetramers (V. E. Mitaksov, S. M. Truscott, L. Lybarger, J. M. Connolly, T. H. Hansen, and D. H. Fremont, submitted for publication) (4).

View larger version (18K): [in this window] [in a new window]   FIGURE 3. T cell recognition of SCT and dtSCT constructs. A, All versions of Kb/OVA SCT are recognized by OT-1 T cells. LM1.8 cells expressing the indicated constructs were tested as targets for cytolytic OT-1 T cells in a 4-h 51Cr release assay. B, 2C T cells recognize the QL9.2m.Ld dtSCT as efficiently as native Ld/QL9. 2C T cells likely recognize Ld bound to endogenous ligand in control cells not fed exogenous QL9 peptide ().

A crucial test of the use of the dtSCT was to measure its capacity to exclude competing peptides. We had previously found that the SCT format without the disulfide trap excluded high-affinity competitor peptides to a great extent (10), 1000 times more effectively than native K^b loaded with endogenous peptides. This exclusion of competitor peptides was enhanced by the Y84A mutation in the SCT that opens the groove and allows for better linker accommodation (11) (V. E. Mitaksov, S. M. Truscott, L. Lybarger, J. M. Connolly, T. H. Hansen, and D. H. Fremont, submitted for publication). To test the dtSCT construct, we incubated cells expressing the dtSCT with increasing amounts of exogenous high-affinity competitor, the K^b-binding SIYR peptide (27). Reactivity of mAb 25-D1.16 was used to specifically monitor the loss of K^b/OVA complexes. In control cells expressing the Y84A SCT, half of the K^b/OVA complexes were displaced by the addition of 125 µM competitor (Fig. 4A, upper left). The cells expressing the OVA.₂m.K^b dtSCT displayed no diminution of reactivity with the 25-D1.16 Ab at the highest concentration tested (500 µM) (Fig. 4A, upper right). Thus, the preponderance of dtSCT molecules retained the OVA peptide moiety in the face of high concentrations of competitor, which was clear evidence that the engineered disulfide bond was present and functioning as expected to lock in the SCT peptide.

View larger version (30K): [in this window] [in a new window]   FIGURE 4. The disulfide trap effectively prevents binding of exogenous competitor peptides. A, Loss of mAb 25-D1.16 reactivity to monitor displacement of OVA or OVAp5Y from Kb SCT constructs. 3KO cells expressing the indicated constructs were incubated with increasing concentrations of exogenous SIYR peptide and analyzed by flow cytometry with Abs specific for Kb (mAb B8-24-3, gray) or Kb/OVA complex (mAb 25-D1.16, black). Mean fluorescence intensity (MFI) at each dose of competitor is plotted as a percentage of the starting signal (no competitor). B, N15 hybridoma activation to detect VSV8 competitor peptide binding to SCT constructs. 3KO cells expressing the indicated constructs, including OVA.2m.Kb SCTs or OVAp5Y.2m.SCTs, were incubated with increasing concentrations of exogenous VSV8 peptide, washed, and cultured with the N15 hybridoma. IL-2 produced upon hybridoma activation was detected measuring CTLL-2 proliferation. N15 activation as a result of VSV8 binding to native Kb (loaded with endogenous peptides in B6/WT3 fibroblasts) is shown for comparison.

A poor binding OVA analog is tethered to the MHC groove using the dtSCT approach

The above results demonstrated that the dtSCT approach can result in stronger binding of the covalently attached peptide. Based on this finding, we hypothesized that a disulfide trap could potentially be exploited to enhance presentation of relatively poor binding peptides. As a first test of this hypothesis we investigated the previously described OVA analog SIINYEKL, or OVAp5Y, which binds poorly to K^b (50, 51, 52) (boldface was used to emphasize that these experiments were conducted with the SIINYEKL and not the SIINFEKL peptide). Howarth et al. (50) showed that the OVAp5Y analog was still recognized in the context of K^b by the 25-D1.16 mAb. Furthermore, they demonstrated that the surface half-life of K^b/OVAp5Y complexes was reduced 3-fold and efficiency of expression >75-fold compared with native K^b/OVA complexes. Therefore, using this OVA analog as a model poor, binding peptide for K^b, we constructed an OVAp5Y.₂m.K^b SCT with and without a disulfide trap (Table I). Although the steady-state cell surface expression level of the OVAp5Y SCT was 10-fold lower than the OVA-based SCT, the surface expression of OVAp5Y SCT was improved 3-fold by the addition of the disulfide trap (data not shown), demonstrating that the disulfide trap facilitates de novo folding and/or increases cell surface stability of SCTs constructed with weak binding peptides. In peptide competition experiments, the OVAp5Y.₂m.K^b Y84A SCT was displaced readily with the SIYR peptide (Fig. 4A, lower left). Staining with the K^b/OVA-specific mAb was half-maximal at 40 µM SIYR compared with 125 µM for the SCT constructed with OVA (Fig. 4A, upper left), which is consistent with its weaker affinity for K^b. However, when the disulfide-trapped OVAp5Y SCT was subjected to the same concentrations of competitor peptide, no loss of reactivity with 25-D1.16 was detected (Fig. 4A, lower right).

The K^b SCT and dtSCT constructed with the weak binding OVA analog were also tested in N15 hybridoma assays (Fig. 4B). Interestingly, when fed the VSV8 peptide, the OVAp5Y SCT Y84A activated the hybridoma somewhat less efficiently, most likely due to its lower K^b surface expression. However, the VSV8 peptide was still effectively excluded from the OVAp5Y.₂m.K^b dtSCT—the N15 hybridoma was not activated even though this construct has 3-fold higher cell surface expression compared with OVAp5Y SCT Y84A. This was a clear demonstration that the engineered disulfide bond is even able to lock in a peptide that binds weakly to the native MHC and thus exclude high-affinity exogenous competitor peptides. The combined findings with OVA- and OVAp5Y-based dtSCTs argue strongly that the engineered disulfide bond is formed properly in most if not all of the cell surface dtSCT molecules.

A second, unstable H-2 complex is secured when expressed as a dtSCT

To test the applicability of the dtSCT technology on an additional class I/peptide complex and to further test its ability to enhance presentation of a complex that turns over rapidly at the cell surface, we used the L^d/QL9 complex. The QL9 peptide (QLSPFPFDL) is derived from an endogenous dehydrogenase and binds to H-2L^d (29, 30, 31), a class I allele that is unique due to its relatively weak association with peptide and ₂m (15, 53). Importantly for this study, the L^d/QL9 complex is relatively unstable based on its limited cell surface half-life (0.5 h) and the difficulty of constructing L^d/QL9 tetramers (T. H. Hansen, unpublished observations). Thus, the L^d/QL9 complex was ideal for testing the stabilizing effects on class I molecules conferred by the single-chain format itself and by the introduction of a disulfide bond. A second advantage of studying the L^d molecule is the ability to directly measure relative amounts of peptide-occupied vs peptide-empty class I conformers at the cell surface with mAb 64-3-7, which specifically detects open (peptide-empty) class I molecules (25, 54, 55, 56). A third reason to construct SCTs of the L^d/QL9 complex was its well-defined reactivity with the 2C TCR (31, 57, 58) and the availability of a soluble recombinant high-affinity 2C-derived mutant TCR designated m6 (34, 35, 36), which is an excellent peptide-specific L^d/QL9 staining reagent for flow cytometry.

Native L^d and QL9.₂m.L^d SCTs with and without the disulfide trap were subcloned individually into a retroviral vector for expression in B6-derived fibroblasts. As a control, a QL9.₂m.L^d SCT with the Y84A mutation was also included. This mutation opens the groove and reduces binding of exogenous peptides (11). As shown in Fig. 5, L^d and the three different L^d/QL9 SCTs were detected at high levels on the cell surface by the mAb 30-5-7, which is specific for folded (peptide-associated) L^d molecules (55). This, combined with the staining pattern of mAb 64-3-7 on each of these transduced cell lines, was especially informative because it provided a relative measure of peptide-induced folding. Native L^d (Fig. 5B) had the highest ratio of open conformers to folded conformers detected by mAb 30-5-7 (open:folded = 1.01), which was an indication of its characteristically weak association with endogenous peptides. Expressing L^d in single-chain format with the QL9 peptide (Fig. 5C) resulted in a lower level of staining with mAb 64-3-7 (open:folded = 0.504) and introducing the Y84A mutation (Fig. 5D) lowered the ratio even further (open:folded = 0.250). By comparison, when the disulfide trap was present in the QL9.₂m.L^d SCT (Fig. 5E), peptide-empty (mAb 64-3-7⁺) conformers were barely detectable (open:folded = 0.012). We concluded from this experiment that the disulfide trap was clearly functional and significantly increased peptide occupancy of this relatively unstable class I/peptide complex.

Introducing two additional cysteine residues, as we had done by engineering the disulfide trap, could theoretically decrease the efficiency of intracellular folding/assembly of these MHC molecules. To test this possibility, both open and folded L^d conformers were precipitated from cell lines expressing native L^d or L^d SCTs with or without the disulfide trap. To compare the relative efficiencies of folding/assembly, immunoprecipitates were treated with endoglycosidase H (EndoH) as shown in Fig. 5F. Clearly, each stage of SCT engineering resulted in progressively fewer EndoH-sensitive (ER-resident) and more EndoH-resistant (post-ER) molecules, demonstrating more rapid assembly and egress from the ER. Furthermore, we quantified the ₂m that coimmunoprecipitated with L^d or L^d SCTs and found that ₂m exchange was eliminated effectively in the dtSCT (Fig. 5G). Thus, introducing the disulfide trap resulted in a dramatic improvement in the efficiency and quality of folding and assembly of class I.

Peptide occupancy determines the half-life of class I molecules at the cell surface (59). L^d, being a relatively poor peptide binder, is thus highly inducible at the cell surface by incubation with exogenous ligands (15). Given this, we reasoned that susceptibility to peptide induction would be informative for comparing the relative peptide occupancy of the L^d/QL9 SCT complexes. As shown in Fig. 6A, culture of cells with exogenous L^d-binding peptide from MCMV (32) increased surface expression of native L^d > 3-fold. Furthermore, exogenous MCMV peptide increased the expression of the QL9.₂m.L^d SCT (3-fold) and, to a lesser extent, the QL9.₂m.L^d Y84A SCT (1.5-fold). The improved refractoriness to exogenous peptide of the SCT with the Y84A mutation was expected based on our previous findings (11). However, the most striking result of this experiment was the constant expression level of the dtSCT at all exogenous peptide concentrations tested. Thus, for relatively unstable pMHC complexes such as L^d/QL9, the disulfide trap clearly renders the SCT more refractory to exogenous peptide binding as monitored by surface stabilization.

We next wanted to extend these findings with L^d/QL9 SCTs with more physiologically relevant TCR-based assays. First, we found that 2C T cells detected target cells expressing QL9.₂m.Ld dtSCT or QL9 peptide-fed targets comparably (Fig. 3B). Then, to detect L^d/QL9 complexes on the cell surface by flow cytometry, we stained these cells with the rTCR m6 (Fig. 5). This high-affinity TCR variant was selected in vitro by yeast display of a library of 2C TCR mutants; its affinity for L^d/QL9 (K_D 9 nM) (35) is >100-fold greater than that of wild-type 2C (K_D 1.5 µM) and thus approaches the affinity of Ab/Ag complexes. Interestingly, m6 bound relatively poorly to the QL9.₂m.L^d SCT (Fig. 5), likely reflecting the fact that residue Y84 in this construct forces the linker extending from the C terminus of the peptide to bulge, hindering m6 binding. Consistent with this conclusion, the SCTs with the Y84A mutation or the disulfide trap have a more relaxed linker (V. E. Mitaksov, S. M. Truscott, L. Lybarger, J. M. Connolly, T. H. Hansen, and D. H. Fremont, submitted for publication) and better detection by rTCR m6 (Fig. 5). In any case, the disulfide-trap form of the QL9.₂m.L^d SCT displayed strong engagement with both the 2C TCR (Fig. 3B) and its derivative, high-affinity rTCR m6 (Fig. 5).

The strong reactivity of m6 TCR for the dtSCT allowed us to determine the extent to which disulfide trapping of the QL9 peptide to L^d excluded competitor peptides relative to native L^d/QL9 complexes. Cells expressing native L^d were fed exogenous QL9 peptide overnight. The next morning, these cells, along with cells expressing each generation of QL9.₂m.L^d SCT, were incubated with increasing concentrations of competitor peptide YPHFMPTNL derived from MCMV pp89 (32), which binds to L^d with similar affinity to QL9 (60). The cells were then stained with recombinant m6 TCR and analyzed by flow cytometry to specifically detect loss of the L^d/QL9 epitope (Fig. 6B). Interestingly, susceptibility to peptide exchange correlated precisely with the levels of cell surface open conformers (staining with mAb 64-3-7; Fig. 5). Native L^d loaded with the QL9 peptide readily bound the MCMV peptide, whereas each generation of L^d SCT was increasingly more resistant to exogenous peptide binding. Even the highest doses of competitor peptide had no effect on the L^d dtSCT. Thus, disulfide bond engineering reformed a normally poor peptide binder into an exceptionally stable MHC molecule.

In summary, dtSCT molecules exhibited proper folding and oxidation in the ER and, upon transport to the cell surface, reacted with MHC-specific Abs and Ag-specific T cells. This disulfide-trap approach generated MHC molecules with remarkable levels of peptide occupancy and resistance to competitor binding. Experiments with both K^b and L^d dtSCTs further demonstrated that placement of the engineered disulfide bond is able to compensate for poor peptide binding to MHC class I. Taken together, these results argue that the dtSCT class I molecules have a disulfide bond that is not only extant but also functional in locking in the SCT peptide, even when the corresponding native pMHC complex has a short half-life.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
In this study, we demonstrate the engineering of a disulfide bond to lock peptide into the MHC class I binding groove. This disulfide trap was introduced into class I molecules expressed as single-chain trimers or SCTs, which have now undergone three distinct generations of molecular engineering. Each of these generations of SCT was recently crystallized and high resolution structures analyzed in collaboration with Dr. Daved Fremont’s lab (V. E. Mitaksov, S. M. Truscott, L. Lybarger, J. M. Connolly, T. H. Hansen, and D. H. Fremont, submitted for publication). These structures verify the native class I conformation of the SCTs, as well as provide an anatomical basis for our biochemical and functional characterizations of each of the three generations of SCTs.

First-generation SCTs are class I MHC molecules expressed in the format: peptide–[G₄S]₃–mature ₂m–[G₄S]₄–mature class I H chain (Fig. 1A). These SCTs exhibited extended cell surface half-life and resistance to exogenous peptide binding, compared with K^b loaded with endogenous peptides (10). However, they were found to be more susceptible to peptide displacement when compared with K^b loaded with OVA peptide (11). These combined observations strongly support the model that the cell surface stability of the SCT and its refractoriness to exogenous peptide binding both reflect the ability of the SCT to quickly rebind the covalently attached peptide. To improve spacer accommodation, the second-generation SCTs included the H chain substitution Y84A, which opened the peptide binding groove where the linker exits (11). As evidence for better linker accommodation, the SCT Y84A displayed improved detection by mAb 25-D1.16 (11) and by the TCR-staining reagent m6 (Fig. 5) compared with SCTs lacking the Y84A mutation.

Although functional SCTs have been constructed from a number of different mouse and human class I pMHC complexes, SCTs constructed with high-affinity peptides are clearly superior. The reason for this is now clear. As noted above, SCTs are dependent upon rebinding of the linker-attached peptide. Consequently, SCTs made with lower affinity peptides have higher steady-state levels of peptide-empty conformers at the cell surface (Fig. 5 and unpublished observations). Thus, further engineering of the SCT was required so that the SCT format would be more applicable to pMHC complexes with lower affinity peptides. Therefore, we began a third round of engineering in the SCT to strategically position a disulfide bond.

The data presented in this article, along with the crystal structure of the dtSCT (V. E. Mitaksov, S. M. Truscott, L. Lybarger, J. M. Connolly, T. H. Hansen, and D. H. Fremont, submitted for publication), confirm that the engineering of the disulfide bond was achieved at H chain position 84 and the second position on the linker extending from the C terminus of the peptide. Migration of the dtSCT in nonreducing SDS-PAGE, as well as the ability of dtSCTs to exclude formidable concentrations of high-affinity competitor peptide, clearly argues in favor of formation of the disulfide bond in cells. Furthermore, the presence of the disulfide bond in the electron density data from dtSCT crystals was unquestionable. Corroborating the native structure revealed in the dtSCT crystal analyses, we show here that dtSCTs of two distinct pMHC complexes are recognized by Abs, TCR reagents, and T cells specific for native pMHC complexes. Due to linker flexibility and use of a conserved position for the H chain cysteine, the disulfide-trap approach presented here will likely befit other H-2 and HLA complexes. Moreover, we have shown that dtSCT construction offers great usage for studying lower affinity pMHC complexes, as weak K^b/peptide and L^d/peptide complexes could both be expressed with a disulfide trap and, more importantly, excluded competitor peptides. As revealed by its crystal structure, our engineering of cysteine residues in the dtSCT provided not only a disulfide bond but also better linker accommodation and fortuitous acquisition of hydrogen bonds for improved peptide anchoring in the F pocket of the dtSCT groove.

Recent applications of SCTs have begun to provide unique insights into our understanding of the complex role of MHC class I molecules in lymphocyte development and function (17, 18, 19). In a seminal study of the role of class I in NK cell development, Kim et al. (17) examined NK cells from mice that expressed the OVA.₂m.K^b SCT as a transgene, but not ₂m or other class I genes. The experiments clearly established that expression of a single MHC class I molecule selectively licenses NK functional activity only in NK precursors with an inhibitory receptor specific for this MHC class I molecule. The SCT also promises to play a pivotal role in studies of CD8 T cell development. To study the development of CD8 T cell in the context of a single MHC class I/peptide complex, we have expressed the OVA.₂m.K^b SCT as a transgene and bred to a K^b-, D^b-, ₂m-deficient background. Importantly, the covalently attached OVA excludes the binding of endogenous peptides, as demonstrated by the strong primary CTL response of T cells from these mice when cultured with cells expressing K^b loaded with endogenous peptides (J. M. Connolly, unpublished observation). However, previous studies of CD4 T cell development made it clear that it is imperative to absolutely ablate endogenous peptide binding to make definitive conclusions about the role of peptide in thymic selection (61, 62, 63). Thus, transgenic mice that express a disulfide-trap OVA.₂m.K^b SCT could be important tools for approaching a key outstanding question in T cell development, i.e., the relationship between the MHC-bound selecting peptide in the thymus vs the activating peptide in periphery.

Beyond basic science, the engineering of the disulfide trap holds great promise for SCT clinical applications. There are already four reports demonstrating that vaccination with plasmid DNA encoding an SCT leads to generation of specific Abs and/or CTL. In the report by Yu et al. (10), DNA encoding the OVA.₂m.K^b SCT was used to vaccinate BALB/c mice. BALB/c mice vaccinated with plasmid encoding SCT were found to generate anti-K^b/OVA Ab, clearly demonstrating that the SCTs are expressed as intact structures by DNA vaccination. Furthermore, in a recent report by Primeau et al. (13), syngeneic immunization of plasmid DNA encoding SCT was found to elicit Ag-specific CTLs. Extending these findings are two exciting recent studies showing that SCT DNA vaccination is highly effective for priming T cells in clinically relevant model systems. In one of these studies, mice doubly transgenic for HLA-A2 and human CD8 were vaccinated with DNA encoding an SCT that included HLA-A2 and a breast cancer-associated peptide derived from mammaglobin (16). This vaccination with SCT DNA was reported by Jaramillo et al. (16) to induce a significant expansion of CTLs capable of specific detection of A2-positive human breast cancer cells. Another recent study by Huang et al. (4) tested SCT DNA vaccination in a mouse model of human papillomavirus (HPV)-induced tumors such as cervical cancer. The HPV oncoprotein E6 is responsible for malignant transformation and is consistently expressed in HPV-associated tumors. Peng et al. (64) identified an immunodominant CTL epitope of the E6 protein that binds to the mouse class I molecule K^b, and this E6 epitope was then incorporated into an SCT (E6p.₂m.K^b). B6 mice were vaccinated with plasmid DNA encoding either the SCT or the intact E6 protein alone. Importantly, DNA vaccination of B6 mice with the SCT elicited high levels of CTL, whereas DNA vaccination with only full-length E6 displayed little response over background. Furthermore, DNA vaccination with SCT protected mice against a lethal tumor challenge whereas vaccination with DNA-encoding E6 or OVA.₂m.K^b SCT did not. These experimental findings represent the first evidence that SCTs can have a significant advantage over protein-based vaccine approaches in a clinically relevant model system. The success of SCTs was credited to the fact they are Ag processing independent, do not have to compete with endogenous peptide, and are more stable at the cell surface due to their covalent nature (4). Most recently, it was shown that SCT-based DNA vaccination generated protection in a model of human ovarian cancer in HLA-A2-transgenic mice (65). Each of these DNA vaccination approaches used first generation SCTs. Although SCT-based DNA vaccines show great promise, they may be improved even further by incorporation of the disulfide trap. Certain vaccines may elicit T cell responses to peptide determinants that bind relatively poorly to the MHC, in which case the use of dtSCTs may have a significant impact on the stability of antigenic determinants, and thus the level of vaccine protection.

We are exploring yet another potential application of our disulfide trap technology in the context of rMHC class I molecules. Rapid dissociation of a peptide from the MHC is a critical limitation in the construction of MHC multimers as FACS staining reagents. Thus, tetramers for several defined CD8 T cell epitopes have been difficult to construct or are very unstable once constructed (66). Furthermore, in vivo applications of rMHC molecules to modulate CD8 responses may require reagents with longer in vivo half-lives and greater thermal stability (67, 68, 69). It was clear from the crystal structure of the dtSCT that an analogous disulfide trap might also be incorporated into soluble, recombinant class I molecules without the SCT linkers. This was done by refolding the class I H chain harboring a Y84C mutation with r₂m and a synthetic peptide having a GC extension. Full characterization of these disulfide-trap pMHC molecules is in progress; however, the approach clearly works for K^b/OVA complexes. The disulfide trap forms properly upon in vitro refolding, and disulfide-trap pMHC tetramers bind T cells specific for the native complex (V. E. Mitaksov, S. M. Truscott, L. Lybarger, J. M. Connolly, T. H. Hansen, and D. H. Fremont, submitted for publication). Using disulfide-trap approaches should enable the construction of stable multimers of lower affinity pMHC complexes and thus make possible more comprehensive analyses of T cell responses to pathogens and tumors.

	Acknowledgments

 
We thank Drs. Lawrence Yu and Michael Miley for indispensable contributions to SCT engineering and characterization, Tina Primeau for expertise in T cell assays, and Nancy Myers for expert technical assistance with cell culture and flow cytometry.

	Disclosures

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S. M. Truscott, L. Lybarger, J. M. Martinko, V. E. Mitaksov, D. M. Kranz, J. M. Connolly, D. H. Fremont, and T. H. Hansen are listed on a pending patent based on single chain format and disulfide traps.

	Footnotes

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

¹ This work was supported by National Institutes of Health Grants AI055849, AI27568, and AI057160.

² Address correspondence and reprint requests to Dr. Ted H. Hansen, Department of Pathology and Immunology, Box 8118, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: hansen{at}pathology.wustl.edu

³ Abbreviations used in this paper: pMHC, peptide/MHC; 3KO, K^b–/–D^b–/–₂m^–/– fibroblast; ₂m, ₂-microglobulin; dtSCT, disulfide-trap single-chain trimer; EndoH, endoglycosidase H; ER, endoplasmic reticulum; [G₄S]_n, n multiples of Gly-Gly-Gly-Gly-Ser; HPV, human papillomavirus; MCMV, murine cytomegalovirus; MFI, mean fluorescence intensity; SCT, single-chain trimer.

Received for publication October 16, 2006. Accepted for publication March 5, 2007.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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