Identification of H-2Kb-, Db- and Dd-binding peptides derived from amino acid sequences of polyoma virus T antigens.
Viral and tumor antigens are presented to cytotoxic T cells (CTL) in the form of short peptides. The peptide antigen is transported to the cell surface in conjunction with molecules encoded by the major histocompatibility complex (MHC). Different methods have recently been described for the analysis of the MHC-class-I binding ability of synthetic peptides. Here, we describe a protocol, based on intact RMA-S cells cultured at 26 degrees C in the presence of synthetic peptides, which gives an allele-specific peptide binding pattern with high resolution. This allowed an analysis of the H-2Kb-, Db- and Dd-binding capacity of a panel of synthetic peptides with amino acid (aa) sequences derived from polyoma (py) virus large-, middle- and small-T (LT, MT and ST) antigens, previously used in immunization experiments against py-virus-induced tumors. Eight single aa mutants and a deletion mutant of one peptide with an ability to bind both to H-2Kb and to Db were also analyzed. We foresee that the present protocol, or variants thereof, may serve as a simple and rapid assay for the systematic screening of the class-I binding ability of large sets of synthetic peptides in vitro. Such analysis may facilitate the search for viral or tumor peptide epitopes that are recognized by CTL.
Reinholdsson-Ljunggren
G
G
Department of Tumor Biology, Karolinska Institutet, Stockholm, Sweden.
Franksson
L
L
Dalianis
T
T
Ljunggren
H G
HG
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006183:H-2 Antigens / Q000378:metabolism*; D064532:Histocompatibility Antigen H-2D; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism*; D017384:Sequence Deletion; D014162:Transfection; D014407:Tumor Cells, Cultured
D000952:Antigens, Polyomavirus Transforming; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D064532:Histocompatibility Antigen H-2D; D010446:Peptide Fragments