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Cutting Edge: Single-Chain Trimers of MHC Class I Molecules Form Stable Structures That Potently Stimulate Antigen-Specific T Cells and B Cells¹

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
We report in this work the expression and characterization of class I molecules expressed as single-chain trimers consisting of an antigenic peptide-spacer-₂-microglobulin-spacer H chain. Our results indicate that these single-chain constructs assemble efficiently, maintain their covalent structure, and are unusually stable at the cell surface. Consequently, these constructs are at least 1000-fold less accessible to exogenous peptide than class I molecules loaded with endogenous peptides, and they are potent simulators of peptide-specific CTL and Abs. Our combined findings suggest that single-chain trimers may have applications as DNA vaccines against virus infection or tumors.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Class I H chains require full assembly with ₂-microglobulin (₂m)³ and peptide to be stably expressed at the cell surface at levels sufficient to induce optimal T cell immunity (1, 2, 3, 4). Class I molecules that fail to complete assembly accumulate in the endoplasmic reticulum (ER) and are targeted for degradation (5). There have been two seminal findings regarding class I assembly in recent years. First, assembly in the ER involves complex interactions with ER proteins that facilitate full class I assembly (6). Second, pathogens and tumors evolve diverse mechanisms to block class I assembly as a means of evading immune detection (7, 8).

As a novel approach to make class I molecules less dependent on chaperone assistance and more resistant to down-regulating mechanisms of viruses, we have produced single-chain class I molecules. There have been two reports of successfully engineering class I molecules by directly linking a peptide ligand to the H chain via a short linker (9, 10), an approach not applicable to most class I/peptide complexes (our unpublished data). Others have reported coupling ₂m to the N terminus of class I molecules with a spacer (11, 12, 13, 14), or covalent linkage of the specific peptide to free ₂m which can then associate with H chains to generate functional trimers (15, 16, 17). Nonetheless, it remains unclear the extent to which the covalent attachment of peptides to ₂m excludes the binding of competing free peptide ligands. We report in this work the first demonstration of linking all three components of class I as a single-chain trimer (SCT), using an approach applicable to all mouse and human class I/peptide complexes tested thus far. Furthermore these SCT efficiently exclude competing peptides and are potent stimulators of Ag-specific T and B cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Mice

B6 (H-2^b), BALB/c (H-2^d), and (C3H x B6)F₁ (H-2^kxb) mice were purchased from Charles River Breeding Laboratories (Wilmington, MA). OT-1 transgenic mice (18) were kindly provided by R. Lorenz (Washington University School of Medicine, St. Louis, MO).

Cell lines, Abs, and peptides

L cell line LM1.8 (H-2^k) (19) was a gift from P. Kourilsky (Institut National de la Santé et de la Recherche Médicale, Institut Pasteur, Paris, France). mAbs used in this study include the following: 64-3-7, which recognizes open (peptide-free) forms of class I molecules tagged with this epitope (20); B8-24-3 (American Type Culture Collection, Manassas, VA), which recognizes folded K^b; and 25D-1.16, which recognizes K^b+ SIINFEKL peptide (21). The OVA-derived peptide (SIINFEKL) (22) and SIYR peptide (SIYRYYGL) (23) were synthesized on a peptide synthesizer (model 432A; PE Applied Biosystems, Foster City, CA).

DNA constructs

Constructs were generated using standard techniques and were confirmed by DNA sequence analysis. The ₂m^b·K^b constructs encode ₂m^b followed by a linker of 15 or 20 residues consisting of (G₄S)_3–4 followed immediately by the mature K^b H chain sequence. The OVA·₂m^b·K^b constructs encode the leader sequence of ₂m^b followed immediately by the SIINFEKL sequence, then a linker of 10 or 15 residues (G₄S)_2–3. This first linker is followed by the mature ₂m^b sequence, the second linker of 15–20 residues, (G₄S)_3–4, then the mature K^b sequence. Constructs were expressed from the pIRES.neo vector (Clontech Laboratories, Palo Alto, CA). The 64-3-7 epitope-tagged K^b mutant (K^bR48Q,R50P) was described previously (24).

Cytotoxic T lymphocytes

The L^d-alloreactive CTL clone, 2C (a gift from H. Eisen, Massachusetts Institute of Technology, Cambridge, MA), was maintained by weekly restimulation with irradiated BALB/c splenocytes as previously described (25). When freshly explanted splenocytes were used, 7.5 x 10⁶ cells were cultured with 3.5 x 10⁶ irradiated splenocytes (2000 rad) for 1 wk in the absence of IL-2 or 3.5 x 10⁵ irradiated L cells (10,000 rad) weekly for 2 wk in the absence of IL-2. Thereafter, 2.5–5 x 10⁵ cells were restimulated weekly with 5 x 10⁶ irradiated splenocytes or 3.5 x 10⁵ irradiated L cells in the presence of 10 U/ml rIL-2. When peptide was included the concentration was 10^-4 M in the absence of IL-2 and 10^-5 M in the presence of IL-2.

Flow cytometry

Viable cells, gated by forward and side scatter, were analyzed on a FACSCalibur (BD Biosciences, San Jose, CA) and data were analyzed using CellQuest software (BD Biosciences). Assays for peptide incubation and turnover of class I were performed as previously reported (24).

Biochemistry

Immunoprecipitations and blots were performed as described (26). Proteins were visualized using ECL reagents (Amersham Pharmacia Biotech, Piscataway, NJ).

DNA vaccination

DNA was prepared and immunizations were conducted according to Corr et al. (27). Briefly, BALB/c mice were injected i.m. in the quadriceps with 100 µg of DNA encoding OVA·₂m^b·K^b. Injections were given twice, separated by a 2-wk interval. Four weeks later mice were bled and tested for Ab production by flow cytometry.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Experimental approach

In this study, several class I SCT constructs were produced. Although they vary in content, each of these SCT consists of the following elements beginning with the amino terminus: the leader sequence of ₂m, the peptide comprising a ligand for the H chain, the first flexible linker of 10 or 15 residues, the mature portion of murine ₂m^b, the second flexible linker of 15 or 20 residues, and, finally, the mature portion of the H chain. To serve as controls, constructs were also made with only ₂m covalently attached to the H chain. These constructs consist of the entire coding region of ₂m^b linked via a 15- or 20-residue spacer to the mature portion of the respective H chain.

Surface expression and functional capabilities of SCTs

Single-chain constructs were initially generated with mouse class I L^d molecules and several different ligands. These SCT constructs resulted in high levels of surface expression and immune recognition by mAb and CTL to L^d (data not shown). However, our characterization of K^b SCTs with SIINFEKL peptide (OVA) and ₂m is the most comprehensive due to well defined OT-1 T cells and mAb 25D-1.16 that specifically recognizes K^b/OVA complexes (18, 21).

To test whether spacer length contributed the immune detection of SCT, a set of OVA·₂m^b·K^b constructs was produced with linkers of different lengths. In addition to varying the linker length, the epitope detected by mAb 64-3-7 was introduced into the K^b H chain to allow detection of peptide empty forms as previously reported (24, 26, 29). As shown in Fig. 1, all of the constructs gave rise to high levels of expression of folded K^b molecules (B8-24-3⁺). However, when examined for the presence of open conformers (64-3-7⁺), only the K^b (Fig. 1A) and ₂m^b(L20)·K^b (Fig. 1B) constructs expressed an appreciable amount, which disappeared with addition of exogenous peptide (data not shown), thus reaffirming their "peptide-empty" nature. In stark contrast, the SCT constructs expressed barely detectable levels of open conformers. Thus, the covalent OVA peptide must be able to occupy the K^b peptide binding groove virtually all the time. Remarkably, the linker combination of (15/20) provided significantly better mAb 25D-1.16 reactivity than the other two combinations. In parallel with the FACS profiles in Fig. 1A, the recognition by OT-1-derived T cells was also the best for these transfectants (Fig. 1B). Thus, increasing the length of the flexible linkers results in improved recognition of the OVA·₂m^b·K^b construct by both the mAb 25D-1.16 and OT-1 T cells. This improved recognition with longer spacers in SCT could reflect better peptide positioning and/or reduced steric hindrance for TCR and Ab interactions.

View larger version (50K): [in this window] [in a new window]   FIGURE 1. Improved Ab and T cell recognition of OVA·2mb·Kb construct by altering linker length. SCT spacer length variants are listed parenthetically, whereby the first and second numbers represent the amino acid length of the spacer between peptide and 2m, and 2m and Kb, respectively. A, Flow cytometric analysis using mAbs B8-24-3 (solid line), 25D-1.16 (dotted line), and 64-3-7 (dashed line) on LM1.8 cells that express the indicated construct. B, Lysis by OT-1 T cells of LM1.8 cells transfected with the indicated construct. Target cells expressing unattached Kb and fed the OVA peptide gave lysis similar to that of the SCT with (15/20) linkers (data not shown). The Kb constructs used were tagged with the 64-3-7 epitope (24 ).

SCT are at least 1000-fold less accessible to loading with exogenous peptide

To assess the stability of the covalent peptide that is anchored in the peptide binding groove, peptide competition assays were performed. To do this, the 2C CTL clone was used because it can recognize K^b in complex with the SIYR peptide (23), which has an affinity for K^b that is comparable to the OVA peptide (31). When ₂m^b·K^b or OVA·₂m^b·K^b transfectants were compared as targets for 2C T cells after overnight incubation with graded doses of SIYR peptide (Fig. 2), the OVA·₂m^b·K^b construct was remarkably resistant to displacement by exogenous SIYR peptide at a concentration as high as 10^-7 M. Contrary to this, there was significant lysis of ₂m^b·K^b transfectants at a concentration as low as 10^-10 M. This finding suggests that the OVA·₂m^b·K^b construct is >1000-fold less accessible to loading by an exogenous peptide of comparable affinity, when compared with the ₂m^b·K^b constructs loaded with endogenous peptides. Thus, the covalent peptide is stably bound in the SCT peptide binding groove.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Resistance of the OVA·2mb·Kb construct to displacement by high-affinity Kb binding peptide. LM1.8-2mb·Kb cells (filled symbols) or LM1.8-OVA·2mb·Kb cells (open symbols) were tested as targets for recognition by 2C T cells. The targets were incubated overnight with the indicated molar concentration of peptide.

The finding that the peptide-binding groove of the OVA·₂m·K^b molecule was relatively inaccessible to exogenous peptide suggested that all of the components of the SCT remain intact at the cell surface (Fig. 2). To examine the integrity of these molecules further, biochemical analyses were performed to compare K^b, ₂m·K^b, and OVA·₂m·K^b. Each of these molecules was immunoprecipitated from respective L cell transfectants and immunoblotted to compare the relative m.w. of all three K^b constructs. As shown in Fig. 3A, mAb 64-3-7 (specific for open H chains) precipitated high levels of K^b but low to undetectable amounts of ₂m·K^b and OVA·₂m·K^b. By contrast, B8-24-3 (specific for folded K^b) was able to precipitate significant amounts of all three constructs. The differential reactivity with these two mAbs demonstrates that the covalent attachments to K^b reduced the levels of open conformers existing at steady-state. In addition, this experiment clearly demonstrated that the ₂m·K^b and OVA·₂m·K^b covalent constructs exhibit a slower migration consistent with their expected m.w. The doublet bands seen with these constructs represent Endo H-sensitive (ER-resident) vs Endo H-resistant (post-ER) synthesis intermediates (data not shown). The low steady-state levels of open SCT conformers was consistent with their rapid ER to Golgi transport when compared with K^b alone as determined in pulse-chase experiments (data not shown).

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Biochemical comparisons of the Kb constructs. For all experiments, the following cells were used: LM1.8·Kb, LM1.8·2m(L20)Kb, and LM1.8·OVA·2m·Kb(15/20). The Kb constructs used were tagged with the 64-3-7 epitope. A, Lysates from equivalent numbers of the indicated cells were precipitated (i.p.) with the mAb listed across the top. Immunoblot of the precipitates, using mAb 64-3-7, is shown. B, Lysates from the indicated cells were immunoprecipitated with mAb 25-D1.16. In some cases, cells were incubated overnight with exogenous OVA peptide before lysis. Precipitates were digested or mock-digested with Endo H before immunoblot with mAb 64-3-7. C, Surface decay following brefeldin A addition (5 µg/ml) was monitored on LM1.8 cells expressing Kb (circles), Kb previously loaded with exogenous OVA peptide (diamonds), or OVA·2mb·Kb (15/20) (squares). Cells were stained with mAb B8-24-3 (open symbols) and mAb 25-D1.16 (filled symbols). Mean fluorescence values were obtained at each time point and plotted as a percentage of the starting signal.

As additional evidence for the integrity of SCT molecules, their surface turnover was compared with that of noncovalently attached K^b molecules with and without overnight incubation with OVA peptide. As shown in Fig. 3C, K^b molecules loaded with endogenous peptides have a half-life of 8 h, whereas K^b molecules loaded with noncovalently bound OVA peptide have a half life of 16 h. Remarkably, the OVA SCT were more stable than the K^b loaded with detached OVA, with no detectable turnover within the 16 h tested. Thus, the integrity of SCT molecules is supported by their 1) resistance to peptide exchange, 2) biochemical detection, and 3) stability at the cell surface.

SCT are immunogenic

The data presented above evince that SCT can assume structures that are recognized by Abs and T cells that were raised originally against native peptide/class I complexes. We next examined the extent to which SCT were capable of stimulating immune responses that would be specific for the native peptide/class I structures. To test the ability of the single-chain class I molecules to generate a T cell response, we compared the ability of LM1.8 (H-2^k) cells expressing OVA·₂m^b·K^b vs ₂m^b·K^b fed exogenous OVA peptide (10^-4 M) to induce K^b/OVA specific T cells in vitro. For this experiment, responder T cells from (C3H(H-2^k) x B6(H-2^b))F₁ mice were used that should respond only to K^b/OVA complexes presented by either OVA·₂m^b·K^b or peptide-fed ₂m^b·K^b. Successful generation of Ag-specific CD8⁺ T cells typically requires in vivo priming, intracellular peptide loading, or Ag-pulsed, purified dendritic cells (22, 32). However, high levels of K^b/OVA-specific lysis were observed after five weekly rounds of in vitro stimulation with the OVA·₂m^b·K^b construct (Fig. 4A). By comparison, after five weekly rounds of stimulation with cells expressing the ₂m^b·K^b construct and fed 10^-4 M continuous OVA peptide, little if any K^b/OVA-specific lysis was observed (Fig. 4A). The high level of occupancy with a single peptide is likely a contributing factor to the strong immunogenicity of SCT as evidenced by their ability to generate peptide-specific T cells after in vitro culture. Another contributing factor could be an impaired interaction with NK inhibitory receptors on NK cells or T cells. Indeed, Chung et al. (14, 33) demonstrated that a linker extending from the C terminus of ₂m to the N terminus of the H chain blocks NK receptor interaction. Likewise, it is also worth considering that SCT could have impaired interaction with CD8 coreceptors. If such were true, T cells resulting from repeated immunization with SCT could be relatively CD8 independent and thus have a higher intrinsic affinity for class I/peptide (34).

View larger version (21K): [in this window] [in a new window]   FIGURE 4. A, Superior immunogenicity of OVA·2mb·Kb (15/20) stimulators over peptide-fed 2mb(L20)·Kb stimulators. Lysis of RMA targets in the absence (open symbols) or continuous presence (filled symbols) of 1 µM OVA peptide by (C3H x B6)F1 effectors after five weekly stimulations with LM1.8-OVA·2mb·etKb (15/20) cells (triangles) or LM1.8-2mb(L20)·Kb cells fed continuous OVA peptide (circles). B, Vaccination of BALB/c mice with DNA encoding OVA·2mb·Kb elicits Abs specific for Kb/OVA complexes. Four weeks after the second DNA injection, sera were obtained and tested for reactivity against LM1.8-Kb cells, with or without overnight incubation in the presence of the Kb binding peptides, OVA, or VSV8. Serum from mouse 3 is shown, as is staining of the same cells with mAb B8-24-3 for comparison.

Collectively, these studies demonstrate that SCT appear to fold correctly when assessed by mAb and T cell recognition. Furthermore, biochemical analyses and peptide competition experiments demonstrate that SCT remain intact, and functional analyses indicate that SCT perform as well as, or in some cases better than, their noncovalent counterparts. We have applied this technology to two murine class I molecules using multiple ligands. In addition, we have evidence that this same approach can be used to generate functional HLA class I SCT (L. Lybarger, Y. Yu, J. Connolly, and T. Hansen, manuscript in preparation). These properties make SCT molecules highly attractive for applications as DNA vaccines or probes of mechanisms of class I assembly in normal and pathogenic conditions.

	Acknowledgments

 
We thank Drs. Ron Germain and Jonathan Yewdell for hybridoma 25D-1.16, Dr. Robin Lorenz for OT-1 transgenic mice, Dr. Herman Eisen for the 2C CTL clone, Dr. Maripat Corr for advice on DNA vaccination, and Nancy Myers for cytofluorometry. Valuable suggestions and critiquing of the manuscript were also provided by Drs. Daved Fremont, David Margulies, and Michael Mage.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI19687, AI42793, AI46553, AI27568, and AI07163.

² Address correspondence and reprint requests to Dr. Ted H. Hansen, Department of Genetics, Washington University School of Medicine, Box 8232, 4566 Scott Avenue, St. Louis, MO 63110. E-mail address: hansen{at}genetics.wustl.edu

³ Abbreviations used in this paper: ₂m, ₂-microglobulin; ER, endoplasmic reticulum; SCT, single-chain trimer.

Received for publication January 23, 2002. Accepted for publication February 7, 2002.

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