ABSTRACT
Over the past two decades, there has been a great interest in the study of HLA-E-restricted αß T cells during bacterial and viral infections, including recently SARS-CoV-2 infection. Phenotyping of these specific HLA-E-restricted T cells requires new tools such as tetramers for rapid cell staining or sorting, as well as for the identification of new peptides capable to bind to the HLA-E pocket. To this aim, we have developed an optimal photosensitive peptide to generate stable HLA-E/pUV complexes allowing high-throughput production of new HLA-E/peptide complexes by peptide exchange. We characterized the UV exchange by ELISA and improved the peptide exchange readout using size exclusion chromatography. This novel approach for complex quantification is indeed very important to perform tetramerization of MHC/peptide complexes with the high quality required for detection of specific T cells. Our approach allows the rapid screening of peptides capable of binding to the non-classical human HLA-E allele, paving the way for the development of new therapeutic approaches based on the detection of HLA-E-restricted T cells.
Subject(s)
Epitopes, T-Lymphocyte/chemistry , Histocompatibility Antigens Class I/chemistry , Major Histocompatibility Complex/immunology , Peptides/chemistry , Amino Acid Sequence , Epitopes, T-Lymphocyte/immunology , High-Throughput Screening Assays , Histocompatibility Antigens Class I/immunology , Humans , Immunologic Techniques , Photochemical Processes , Protein Binding , Protein Conformation , T-Lymphocytes, Cytotoxic/immunology , HLA-E AntigensABSTRACT
BACKGROUND: Commercial hydrolysed diets are used for the diagnosis of food allergy in dogs. The cleaved parent proteins are presumed to be too small to elicit an allergic response by reacting with allergen-specific immunoglobin E (IgE). OBJECTIVES: To evaluate three commercial hydrolysed dog diets for proteins. ANIMALS: Sera were collected from dogs with suspected food allergy. METHODS: Two batches of each hydrolysed diet were examined by electrophoresis and visualized by Coomassie blue, silver nitrate staining and IgE immunoblotting. RESULTS: From two to five proteins, ranging from 21 to 67 kDa, were detected in all three diets evaluated. Circulating IgE antibodies targeting these proteins were detected by immunoblotting of dog sera. Six different carbohydrate proteins were identified by mass spectrometry; maize/potato granule-bound starch synthase-1, soybean glycinin, soybean ß-conglycinin α chain, potato aspartic protease inhibitor, rice glutelin type B1 and soybean sucrose-binding protein. Four of these proteins have been described as allergens in humans. CONCLUSIONS: Some commercial hydrolysed diets contain carbohydrate proteins. Some dogs have circulating IgE antibodies targeting these proteins. The clinical significance of these findings is unknown.
