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

Abstract

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.M HC Class I molecules are heterotrimeric complexes consisting of an invariant light chain called ␤2-microglobulin (␤2m), a polymorphic heavy chain (HC) and an Ϸ8-to 11-aa peptide ligand. These peptide-MHC (pMHC) complexes are recognized by the T cell receptor (TCR) of CD8 ϩ T cells in a peptide-specific fashion, and this interaction forms the molecular basis of antigen recognition by CD8 ϩ T cells. In the past decade, the mapping of pathogen-specific and autoimmune-or cancer-associated T cell epitopes has been a major driving force in the development of assay systems for immunomonitoring. In addition, knowledge of such T cell epitopes forms a cornerstone in the development of vaccine-based or adoptive T cell therapies. As a first step in the mapping of disease-associated T cell epitopes, peptide fragments of disease-associated proteomes may be analyzed for binding to MHC molecules of interest, and subsequent assays can then be used to determine whether T cell reactivity against such pMHC complexes does occur. As demonstrated in a landmark study by Altman and colleagues (1), such antigen-specific T cell reactivity can efficiently be detected by the staining of T cell populations with recombinant fluorescent multimeric MHC molecules.There is an increasing interest in the development of assay systems, such as MHC-based microarrays, that can monitor a multitude of T cell responses in parallel (2-4). Unfortunately, current technology does not allow for the high-throughput generation of different pMHC complexes, thereby limiting the utility of these techniques. Specifically, because MHC class I complexes that are devoid of peptide are markedly unstable (5, 6), current production processes for recombinant MHC complexes require inclusion of a specific T cell epi...

Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules

“…However, the combination of both precursors through a splicing reaction may form a product that then displays high affinity for MHC class I. Using this FP assay (26,27), the HLA-A*0201 binding affinities of reaction mixtures containing various C-terminal ligation partners were measured to determine the relative ligation efficiency of each precursor. A series of C-terminal precursors XLPSV or KXPSV or KLXSV (in which X is any proteogenic amino acid) were each incubated with purified 20S proteasome in the presence of the N-terminal precursor YLDW-SY to allow formation of the transpeptidation products [ (Fig.…”

“…After 30 min irradiation, the plate was sealed with thermowell sealing tape (Corning) and incubated at room temperature for 4 or 24 h, allowing cleaved peptide fragments to dissociate and to be exchanged for competitor and/or tracer peptide. Subsequently, FP measurements were performed as described previoiusly (27). Data were analyzed using GraphPad Prism software (GraphPad).…”

UV-Induced Ligand Exchange in MHC Class I Protein Crystals

“…[15][16][17] Here we present a method that is also based on light-induced chemical reactions, but with the aim to generate structures of novel complexes within the existing crystal. This method is based on the use of peptide tools we have recently described (Figure 1), [18][19][20] and makes pMHC complexes amenable to HTP X-ray crystallographic analysis.…”

Class I Major Histocompatibility Complexes Loaded by a Periodate Trigger

“…human MHC class I HLA-A1, -A2, -A3, -A11 and -B7, [8][9][10] . In subsequent work, this technology has proven useful for the identification of human and murine T cell epitopes in Chlamydia, 11 murine gamma herpes virus, 12 Toxoplasma gondii 13 and melanoma-associated antigens (ref 10 and Reker-Hadrup et al…”

“…As a first generation of such conditional MHC ligands we have designed photocaged MHC ligands that can be cleaved by a UV trigger in the MHC bound state under conditions that do not affect the integrity of the MHC structure ( Fig. 1a and b) (Celie et al, manuscript submitted) [8][9][10] . MHC class I complexes occupied with such photocaged ligands can be produced and purified using standard strategies.…”