Interactions between Immunogenic Peptides and MHC Proteins
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Exhausted CD8 T (Tex) cells are a distinct cell lineage that arise during chronic infections and cancers in animal models and humans. Tex cells are characterized by progressive loss of effector functions, high and sustained inhibitory receptor expression, ...Read MoreKeywords:
Polyproline helix
Receptor–ligand kinetics
Proteolysis
MHC restriction
CD74
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The binding kinetics between cell surface receptors and extracellular biomolecules is critical to all intracellular and intercellular activity. Modeling and prediction of receptor-mediated cell functions are facilitated by measurement of the binding properties on whole cells, ideally indicating the subcellular locations or cytoskeletal associations that may affect the function of bound receptors. This dual need is particularly acute vis à vis ligand engineering and clinical applications of antibodies to neutralize pathological processes. Here, we map individual receptors and determine whole-cell binding kinetics by means of functionalized force imaging, enabled by scanning probe microscopy and molecular force spectroscopy of intact cells with biomolecule-conjugated mechanical probes. We quantify the number, distribution, and association/dissociation rate constants of vascular endothelial growth factor receptor-2 with respect to a monoclonal antibody on both living and fixed human microvascular endothelial cells. This general approach to direct receptor imaging simultaneously quantifies both the binding kinetics and the nonuniform distribution of these receptors with respect to the underlying cytoskeleton, providing spatiotemporal visualization of cell surface dynamics that regulate receptor-mediated behavior.
Receptor–ligand kinetics
Cell surface receptor
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BackgroundCytotoxic T Lymphocytes (CTL) recognize complexes of peptide ligands and Major Histocompatibility Complex (MHC) class I molecules presented at the surface of Antigen Presenting Cells (APC). Detection and isolation of CTL's are of importance for research on CTL immunity, and development of vaccines and adoptive immune therapy. Peptide-MHC tetramers have become important reagents for detection and enumeration of specific CTL's. Conventional peptide-MHC-tetramer production involves recombinant MHC production, in vitro refolding, biotinylation and tetramerization; each step followed by various biochemical steps such as chromatographic purification, concentration etc. Such cumbersome production protocols have limited dissemination and restricted availability of peptide-MHC tetramers effectively precluding large-scale screening strategies involving many different peptide-MHC tetramers.Methodology/Principal FindingsWe have developed an approach whereby any given tetramer specificity can be produced within 2 days with very limited effort and hands-on time. The strategy is based on the isolation of correctly oxidized, in vivo biotinylated recombinant MHC I heavy chain (HC). Such biotinylated MHC I HC molecules can be refolded in vitro, tetramerized with streptavidin, and used for specific T cell staining-all in a one-pot reaction without any intervening purification steps.Conclusions/SignificanceWe have developed an efficient "one-pot, mix-and-read" strategy for peptide-MHC tetramer generation, and demonstrated specific T cell straining comparable to a commercially available MHC-tetramer. Here, seven peptide-MHC tetramers representing four different human MHC (HLA) class I proteins have been generated. The technique should be readily extendable to any binding peptide and pre-biotinylated MHC (at this time we have over 40 different pre-biotinylated HLA proteins). It is simple, robust, and versatile technique with a very broad application potential as it can be adapted both to small- and large-scale production of one or many different peptide-MHC tetramers for T cell isolation, or epitope screening.
Tetramer
MHC restriction
Streptavidin
CTL*
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MHC restriction
Antigen processing
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We have been interested in the final stage of antigen processing and presentation by MHC class II molecules. More precisely, the main aim of this project was to examine the cellular requirements for the binding of exogenous peptides to the mouse B lymphoma cell line, A20. In order to detect directly specific peptide/MHC class II complexes on A20 we have immunoselected a low class II expressor variant from A20. This cell line, called UV, was characterized biochemically and functionally. MHC Class II expression of UV is reduced to 5% of the level of the original cell line, due to a transcriptional deficiency, and is unable to present either antigen or peptide to specific T cell hybridoma. Moreover it has a decreased level of membrane MHC class I molecules. Using several negative control cell lines including UV, we have set up a two-step fluorescent peptide binding assay to A20, consisting in a first incubation with a biotinylated peptide and a second incubation in presence of a fluoresceinated derivative of streptavidin. This assay was validated in several ways. Using this assay, we have analysed the effect of various pharmacological and physical agents on peptide binding. Our results show that most of the peptide binding does not require either newly synthesised or recycling MHC class II molecules. Moreover, it still occurs in the absence of metabolic energy. We also showed that peptide binding depends on the membrane fluidity. Unexpectedly, peptide/MHC class II complexes have a remarkably low half-life as compared to that of soluble complexes. A cellular model for the binding of exogenous peptides to A20 is discussed. The secondary aim of this thesis was to examine the molecular characteristics of MHC class II molecules in relation to peptide binding, using a pan el of mutant MHC class II transfectants and the fluorescent peptide binding assay. The results of the analysis of mutations affecting the peptide binding site are consistent with the structural model of class II. We also showed the necessity of a close packing of the transmembrane domains of class II α and β chains for efficient peptide binding.
MHC restriction
Streptavidin
Antigen processing
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MHC class I molecules bind short peptides for presentation to CD8+ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class I a molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1c, which carries unusual key residues at both ends of the groove, and RT1.Aa which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7 – 15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-Kb, 8 – 13-mer and H2-Db, 9 – 15-mer peptides). The rat MHC class Ia molecule, RT1-Aa, revealed a preference for 9 – 15-mer peptides, whereas RT1-A1c showed a more stringent preference for 9 – 12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.
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Abstract: Many different assays for measuring peptide–MHC interactions have been suggested over the years. Yet, there is no generally accepted standard method available. We have recently generated preoxidized recombinant MHC class I molecules (MHC‐I) which can be purified to homogeneity under denaturing conditions (i.e., in the absence of any contaminating peptides). Such denatured MHC‐I molecules are functional equivalents of “empty molecules”. When diluted into aqueous buffer containing beta‐2 microglobulin (β 2 m) and the appropriate peptide, they fold rapidly and efficiently in an entirely peptide dependent manner. Here, we exploit the availability of these molecules to generate a quantitative ELISA–based assay capable of measuring the affinity of the interaction between peptide and MHC‐I. This assay is simple and sensitive, and one can easily envisage that the necessary reagents, standards and protocols could be made generally available to the scientific community.
Beta-2 microglobulin
MHC restriction
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CTL*
MHC restriction
Streptavidin
Avidity
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