The first step of peptide selection in antigen presentation by MHC class I molecules.

MHC class I molecules present a variable but limited repertoire of antigenic peptides for T-cell recognition. Understanding how peptide selection is achieved requires mechanistic insights into the interactions between the MHC I and candidate peptides. We find that, at first encounter, MHC I H-2K(b) considers a wide range of peptides, including those with expanded N termini and unfitting anchor residues. Discrimination occurs in the second step, when noncanonical peptides dissociate with faster exchange rates. This second step exhibits remarkable temperature sensitivity, as illustrated by numerous noncanonical peptides presented by H-2K(b) in cells cultured at 26 °C relative to 37 °C. Crystallographic analyses of H-2K(b)-peptide complexes suggest that a conformational adaptation of H-2K(b) drives the decisive step in peptide selection. We propose that MHC class I molecules consider initially a large peptide pool, subsequently refined by a temperature-sensitive induced-fit mechanism to retain the canonical peptide repertoire.

Altered peptide ligands revisited: vaccine design through chemically modified HLA-A2-restricted T cell epitopes.

Virus or tumor Ag-derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation of nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligands (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A*0201 revealed the molecular interactions likely responsible for improved binding. The best CPLs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients.

Development of a hypersensitive periodate-cleavable amino acid that is methionine- and disulfide-compatible and its application in MHC exchange reagents for T cell characterisation.

Incorporation of cleavable linkers into peptides and proteins is of particular value in the study of biological processes. Here we describe the synthesis of a cleavable linker that is hypersensitive to oxidative cleavage as the result of the periodate reactivity of a vicinal amino alcohol moiety. Two strategies directed towards the synthesis of a building block suitable for solid-phase peptide synthesis were developed: a chemoenzymatic route, involving L-threonine aldolase, and an enantioselective chemical route; these led to α,γ-diamino-ß-hydroxybutanoic acids in diastereoisomerically mixed and enantiopure forms, respectively. Incorporation of the 1,2-amino alcohol linker into the backbone of a peptide generated a conditional peptide that was rapidly cleaved at very low concentrations of sodium periodate. This cleavable peptide ligand was applied in the generation of MHC exchange reagents for the detection of antigen-specific T cells in peripheral blood cells. The extremely low concentration of periodate required to trigger MHC peptide exchange allowed the co-oxidation of methionine and disulfide residues to be avoided. Conditional MHC reagents hypersensitive to periodate can now be applied without limitations when UV irradiation is undesired or less practical.

Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7.

Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic CD8(+) T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separate in vitro MHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals.

Improved single-chain transactivators of the Tet-On gene expression system.

BACKGROUND: The Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes. Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). To allow the independent regulation of multiple genes, novel Tet systems are being developed that respond to different effectors and bind to different tetO sites. To prevent heterodimerization when multiple Tet systems are expressed in the same cell, single-chain variants of the transactivators have been constructed. Unfortunately, the activity of the single-chain rtTA (sc-rtTA) is reduced when compared with the regular rtTA, which might limit its application. RESULTS: We recently identified amino acid substitutions in rtTA that greatly improved the transcriptional activity and doxycycline-sensitivity of the protein. To test whether we can similarly improve other TetR-based gene regulation systems, we introduced these mutations into tTA and sc-rtTA. Whereas none of the tested mutations improved tTA activity, they did significantly enhance sc-rtTA activity. We thus generated a novel sc-rtTA variant that is almost as active and dox-sensitive as the regular dimeric rtTA. This variant was also less sensitive to interference by co-expressed TetR-based tTS repressor protein and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used. CONCLUSION: We developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used.

Chemical Modification of Influenza CD8+ T-Cell Epitopes Enhances Their Immunogenicity Regardless of Immunodominance.

T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines.

Technologies for MHC class I immunoproteomics.

T cell epitopes are peptides, for instance derived from foreign, mutated or overexpressed proteins, that are displayed by MHC molecules on the cell surface and that are recognized by T lymphocytes. Knowledge of the identity of epitopes displayed by MHC molecules is of high value for diagnostic purposes and for the development of prophylactic and therapeutic immunotherapy regimens. Here we review key techniques in MHC class I epitope definition and we discuss recent developments in epitope discovery and their implications. Developments in epitope discovery strategies should ultimately lead to the definition of the MHC-associated peptidome.

Delivery of 2-5A cargo into living cells using the Tat cell penetrating peptide: 2-5A-tat.

2',5'-Oligoadenylate tetramer (2-5A) has been chemically conjugated to short HIV-1 Tat peptides to provide 2-5A-tat chimeras. Two different convergent synthetic approaches have been employed to provide such 2-5A-tat bioconjugates. One involved generation of a bioconjugate through reaction of a cysteine terminated Tat peptide with a alpha-chloroacetyl derivative of 2-5A. The second synthetic strategy was based upon a cycloaddition reaction of an azide derivative of 2-5A with a Tat peptide bearing an alkyne function. Either bioconjugate of 2-5A-tat was able to activate human RNase L. The union of 2-5A and Tat peptide provided an RNase L-active chimeric nucleopeptide with the ability to be taken up by cells by virtue of the Tat peptide and to activate RNase L in intact cells. This strategy provides a valuable vehicle for the entry of the charged 2-5A molecule into cells and may provide a means for targeted destruction of HIV RNA in vivo.